EA046580B1 - CATIONIC NEUROTOXINS - Google Patents

Description

The present invention relates to engineered clostridia toxins including at least one amino acid modification, as well as the use of such engineered clostridia toxins in medicine and therapy.

Bacteria of the genus Clostridia produce highly active and specific protein toxins that have the ability to damage neurons and other cells into which they penetrate. Examples of such clostridial toxins are the neurotoxins produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes A-G, as well as the neurotoxins produced by C. baratii and C. butyricum.

Some of the best known active toxins are clostridial toxins. For example, the median lethal dose (LD50) of botulinum neurotoxins in mice ranges from 0.5 to 5 ng/kg, depending on the serotype. Tetanus and botulinum toxins act by inhibiting the function of damaged neurons, and in particular the release of specific neurotransmitters. Botulinum toxins act at the neuromuscular junction and inhibit cholinergic signaling in the peripheral nervous system, while tetanus toxins act in the central nervous system.

By their nature, clostridial toxins are synthesized as a single-chain polypeptide that undergoes post-translational modification through proteolytic cleavage to form two polypeptide chains linked to each other by a disulfide bond. Cleavage occurs at a specific cleavage site, often called an activation site, which is located between cysteine residues forming an interchain disulfide bond. As a result, a double-stranded molecule is formed, which is the active form of the toxin. The double-stranded molecule consists of a heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and a light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain contains an N-terminal translocation component (HN domain) and a C-terminal targeting component ( _HC domain). The cleavage site is located by the L chain and translocation domain components. After binding of the HC domain to its target neuron and internalization of the bound toxin into the cell via the endosome, the HN domain translocates the L chain across the endosomal membrane and the cytosol, and this L chain provides protease function (also known as a noncytotoxic protease).

Noncytotoxic 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 comes from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide susceptibility factor. SNARE proteins are integral intracellular vesicle proteins that secrete molecules by transporting the vesicle out of the cell. The protease function is zinc-dependent endopeptidase activity, where such protease function provides a high degree of substrate specificity for SNARE proteins. Accordingly, the noncytotoxic protease, once it enters the desired target cell, has the ability to inhibit cellular secretion from the target cell. Clostridia toxin L-chain proteases are noncytotoxic proteases that cleave SNARE proteins.

Because SNARE proteins are widely distributed in nature, clostridial toxins, such as botulinum toxin, have been successfully used in a wide range of therapies.

For example, the authors refer to the publication William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004), which describes the use of clostridial toxins for various therapeutic and cosmetic purposes, for example, toxins such as botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F and BoNT/G and tetanus neutoxin (TeNT) used to inhibit neuronal transmission, for example, the use of BOTOX™, which is currently used as a therapeutic agent for the treatment of diseases such as: achalasia, adult spasms, anal fissures, back pain, blepharospasm, bruxism, cervical dystonia, essential tremor, glabellar folds or hyperkinetic facial folds, headache , hemifacial spasms, overactive bladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis, dancing eye disease, nasal wrinkles, spasmodic dysphonia, strabismus and nervous disorder VII. In addition, the therapeutic use of clostridial toxins for the treatment of neuromuscular disorders has been described (see US Pat. No. 6,872,397); for the treatment of diseases of the uterus (see US application 2004/0175399); for the treatment of ulcers and gastroesophageal reflux (see US application 2004/0086531); for the treatment of dystonia (see US patent 6319505); for the treatment of eye diseases (see US application 2004/0234532); for the treatment of blepharospasm (see US application 2004/0151740); for the treatment of strabismus (see US application 2004/0126396); for pain relief (see US patents 6869610, 6641820, 6464986 and 6113915); for the treatment of fibromyalgia (see US patent 6623742, US application 2004/0062776); for the relief of low back pain (see US application 2004/0037852); for the treatment of muscle damage (see US patent 6423319); for relief of headaches in the sinus region (see US patent 6838434); for the relief of tension headaches (see US patent 6776992); for relief of headaches (see US patent 6458365); for migraine headache (see US patent

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5714469); for the treatment of cardiovascular diseases (see US patent 6767544); for the treatment of nervous disorders such as Parkinson's disease (see US patents 6620415 and 6306403); for the treatment of neuropsychiatric disorders (see US applications 2004/0180061 and 2003/0211121); for the treatment of endocrine diseases (see US patent 6827931); for the treatment of thyroid diseases (see US patent 6740321); for the treatment of diseases of the sweat glands caused by the action of cholinergic receptors (see US patent 6683049); for the treatment of diabetes (see US patents 6337075 and 6416765); for the treatment of pancreatic disease (see US patents 6261572 and 6143306); for the treatment of cancer, such as bone cancer (see US patents 6565870, 6368605 and 6139845, and US application 2005/0031648); for the treatment of ear diseases (see US patents 6358926 and 6265379); for the treatment of autonomic disorders such as gastrointestinal muscle disorders and other smooth muscle dysfunctions (see US Pat. No. 5,437,291); for the treatment of skin lesions associated with cutaneous cell proliferating diseases (see US Pat. No. 5,670,484); for the treatment of neurogenic inflammatory disorders (see US patent 6063768); to reduce hair loss and stimulate hair growth (see US patent 6299893); for the treatment of scoliosis (see US patent 6358917); to reduce appetite (see US application 2004/40253274); for the treatment and removal of teeth (see US application 2004/0115139); for the treatment of neuromuscular disorders and pathologies (see US application 2002/0010138); for the treatment of various disorders and conditions and for the management of pain caused by such disorders and conditions (see US application 2004/0013692); for the treatment of conditions caused by mucosal hypersecretion, such as asthma and COPD (see application WO 00/10598); and for the treatment of conditions other than nervous disorders such as inflammation, endocrine disorders, exocrine disorders, immunological disorders, cardiovascular diseases, bone diseases (see application WO 01/21213). All of the above publications are incorporated herein by reference in their entirety.

It is envisaged that noncytotoxic proteases, such as clostridial toxins (eg, BoNT and TeNT), can be used for the therapeutic treatment of humans and other mammals, as well as in cosmetics, and in addition, these proteases can be used to treat a wide range of diseases and disorders , which may be treatable due to the beneficial properties of these toxins.

To avoid systemic neurological effects, in many treatments using clostridia toxins, therapeutic clostridia toxins are directly administered to a given target site (such as target tissue). Therapy administered by administering clostridial toxins has problems with the spread of the toxin beyond the injection site and into surrounding tissue or the bloodstream. It is clear that diffusion of the toxin from the target tissue can lead to undesirable side effects, which, in exceptional cases, may pose a threat to the patient's life. This particularly applies to the use of therapeutic clostridial toxins (such as BoNT therapeutics) in high doses, concentrations and injection volumes. Side effects associated with problems encountered with the administration of commercially available BoNT/A therapeutics include asthenia, general muscle weakness, diplopia, blepharoptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and difficulty breathing. Difficulty in swallowing and breathing can be life-threatening and has been reported to be fatal due to the spread of cytotoxic toxins.

Therefore, it is necessary to obtain clostridial toxins that, unlike known clostridial toxins, would have an increased ability to be retained in the tissue at the site of their introduction and a reduced ability to diffuse from the site of administration.

The present invention solves the above problems by constructing the clostridia toxins claimed in the present invention.

In one aspect, the present invention relates to an engineered clostridia toxin comprising at least one amino acid modification, wherein the at least one amino acid modification causes the isoelectric point (pI) of the engineered clostridia toxin to increase 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) units higher than the pI of any another identical clostridial toxin that does not contain at least one specified amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the constructed clostridia toxin to a value that is at least 0.4 units higher than the pI of any other identical clostridia toxin not containing at least one the specified amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the constructed clostridia toxin to a value that is at least 0.5 units higher than the pI of any other identical clostridia toxin not containing at least one the specified amino acid modification. In one embodiment of the invention, at least one specified amino acid modification increases the pI of the constructed clostridial toxin to a value that is at least

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0.6 units higher than the pI of any other identical clostridial toxin that does not contain at least one specified amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the constructed clostridia toxin to a value that is at least 0.8 units higher than the pI of any other identical clostridia toxin not containing at least one the specified amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the constructed clostridia toxin to a value that is at least 1 unit higher than the pI of any other identical clostridia toxin not containing at least one amino acid modification.

In one aspect, the present invention provides an engineered clostridial toxin comprising at least one amino acid modification, wherein the at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least one unit The pI is higher than the pI of any other identical clostridial toxin that does not contain at least one specified amino acid modification.

In some embodiments, the engineered clostridium toxin contains at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 8 0 amino acid modifications.

In some embodiments, the at least one amino acid modification increases the pI of the engineered clostridia toxin to a value that is at least 2, 3, 4, or 5 pI units higher than the pI of any other identical clostridia toxin that does not contain at least one specified amino acid modification.

The present inventors have discovered that by increasing the pI of the clostridium toxin, for example, by at least 0.2 pI unit or 0.5 pI unit or one pI unit (by introducing at least one amino acid modification into the clostridium toxin protein) , the ability of the resulting engineered clostridial toxin to be retained in tissue is preferentially increased, and its ability to diffuse from injection sites is reduced, while maintaining the ability of target cells to bind to the target SNARE protein(s), as well as the ability to transport this(their ) protein(s) and its (their) breakdown. Thus, the spread of the clostridia toxin from the injection site is significantly reduced compared to the spread of any other identical clostridia toxin that does not contain at least one of the specified amino acid modifications.

The engineered clostridial toxins of the invention can be used in any of the treatment methods described above, and these toxins, compared with known therapeutic clostridial toxins, have substantially fewer or no side effects.

The increased ability of the engineered clostridial toxins according to the invention to remain in tissue also provides their increased activity and/or longer duration of action, and therefore, these toxins, in comparison with known therapeutic clostridial toxins, can be used in lower doses (or in higher doses without any other side effects), indicating their additional benefits.

As discussed in detail below, the increase in pI achieved by introducing at least one amino acid modification means that the engineered clostridial toxin of the invention has, at a given pH, a net charge greater than the net charge of any other identical clostridial toxin not containing at least at least one specified amino acid modification. Without wishing to be bound by any particular theory, we merely note that this increased positive charge provides the engineered clostridial toxin of the invention with the ability to be retained in the tissue at the site of administration for a longer period of time due to favorable electrostatic interactions between the engineered clostridial toxin and anionic extracellular components (such as cell membranes and heparin sulfate-based proteoglycans) at the injection site. Such favorable electrostatic interactions reduce the ability of the engineered clostridial toxin to diffuse from the injection site and thereby increase tissue retention time.

For example, the increased tissue retention capacity of the engineered clostridial toxin of the invention allows (i) higher doses of the toxin to be administered to individual muscles, such as the sternocleidomastoid muscles, without fear of spreading the toxin to areas adjacent to the neck muscles , which leads to difficulty swallowing, and (ii) higher total doses (to all muscles) per course of therapy, without the fear of spreading this toxin into the bloodstream, which causes systemic side effects such as difficulty breathing. For patients, the advantage of this toxin administration is that it provides more effective treatment for diseases of large muscles, such as the sternocleidomastoid muscles, and

- 3 046580 allows for injections into several different muscles in each individual course of treatment, and also, thanks to the administration of higher doses, to carry out, if possible, a longer effective treatment (for a longer period of time before repeating the treatment cycle, if it includes the need arises).

In one embodiment, the engineered clostridial toxin used in the present invention has a positive net charge (eg, if the engineered clostridial toxin is localized at the desired site of tissue administration).

The isoelectric point (pI) is a specific parameter of a given protein. As is well known in the art, proteins are composed of a specific amino acid sequence (also called protein amino acid residues). Each amino acid in the standard set of twenty amino acids has a specific side chain (or R group), meaning that each amino acid residue in a protein has different chemical properties, such as charge properties and hydrophobicity. These properties can be influenced by the protein's chemical environment, such as its temperature and pH. The overall chemical properties of a protein depend on the sum of these various factors.

Some amino acid residues (detailed below) have ionizable side chains that can carry a specific electrical charge depending on the pH of the environment. Whether a side chain is charged or uncharged at a given pH depends on the pKa of the relevant ionizable group, where pKa is the negative logarithm of the acid dissociation constant (Ka) for the particular proton originating from the conjugate base.

Thus, for example, acidic residues such as aspartic acid and glutamic acid residues have side chain carboxyl groups with pKa values of approximately 4.1 (the exact pKa values may depend on temperature, ionic strength, and the microenvironment of the ionizable group).

Thus, these side chains have a negative charge at pH 7.4 (often called physiological pH). At low pH values, these side chains become protonated and lose their charge.

In contrast, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values of approximately 10-12. Therefore, these side chains have a positive charge at pH 7.4. Such side chains become deprotonated and lose their charge at high pH values.

Therefore, the net charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and on the degree of their accessibility on the protein surface) and on the pH of the environment. Changing the pH of the environment leads to a change in the overall charge of the protein. In accordance with this, for each protein there is a certain pH at which the magnitude of the positive and negative charges are equal, and therefore the protein is uncharged. This parameter is known as the isoelectric point (pI). The isoelectric point is a standard parameter that is used in protein biochemistry and is known to those skilled in the art.

Therefore, the isoelectric point (pI) is defined as the pH value at which a protein has a net charge of zero. An increase in pI means that the protein requires a higher pH to achieve a net charge of zero. Thus, an increase in pI means an increase in the net positive charge of the protein at a given pH. Conversely, a decrease in pI means that in this case, the protein requires a lower pH value to achieve a net charge of zero. Thus, a decrease in pI means a decrease in the net positive charge of the protein at a given pH.

Methods for determining protein pI are described in the literature and are known to specialists in the field. For example, pI can be calculated from the average pKa values for each amino acid present in a protein. Alternatively, pI can be determined experimentally by the isoelectric focusing method. This method uses electrophoresis to separate proteins based on their pI. Isoelectric focusing is usually carried out using a gel having a constant pH gradient. After applying an electric field, proteins migrate in the direction of the pH gradient until the pH reaches a value at which the net charge is zero, a value called the pI of the protein.

The isoelectric point of a protein can be increased or decreased by changing the number of basic and/or acidic groups present on the surface of the protein. This can be achieved by modifying one or more amino acids of 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 are discussed in more detail below.

Native (unmodified) clostridia toxins have a pI of approximately 5-6. Thus, at pH 7.4, native botulinum toxins have a net negative charge. For example, the pI for BoNT/A is 6.4, and the BoNT/A molecule has a net charge at pH 7.4 of 8. These pI values are calculated as described above.

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Table 1

Clostridium toxin P.I. BoNT/A 6, 4 BoNT/B 5, 3 BoNT/Ci 5, 5 BoNT/D 5, 5 BoNT/E 6, 0 BoNT/F 5, 6 BoNT/G 5, 2 TeNT 5, 8

As described above, in one embodiment of the invention, the engineered clostridial toxin of the invention includes at least one amino acid modification, wherein the at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of any other identical clostridial toxin that does not contain at least one specified amino acid modification.

Thus, in the context of the present invention, an increase in the pI of the engineered clostridial toxin BoNT/A by 0.2 units means an increase in the pI from 6.4 to 6.6.

As described above, in one embodiment of the invention, the engineered clostridial toxin of the invention includes at least one amino acid modification, wherein the at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least one pI unit higher than the pI of any other identical clostridial toxin that does not contain at least one specified amino acid modification.

Thus, in the context of the present invention, an increase in the pI of the engineered clostridial toxin BoNT/A by 1 unit means an increase in the pI from 6.4 to 7.4.

In one embodiment, the at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least two pI units higher than the pI of any other identical clostridial toxin that does not contain at least one specified amino acid modification.

In one embodiment, the at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridia toxin to a value that is at least 2-5 pI units higher than the pI of any other identical clostridia toxin, not containing at least one specified amino acid modification.

In one embodiment, the engineered clostridium toxin has a pI of at least 6 (eg, at least 6, at least 7, at least 8, or at least 9).

In one embodiment of the invention, the engineered clostridium toxin has a pI of at least 7.

In one embodiment of the invention, the engineered clostridial toxin has a pI of 6 to 10 (eg, 7 to 9 or 8 to 9).

As discussed above, the engineered clostridial toxins of the invention have an increased ability to be retained in tissue, which also provides them with increased potency and/or longer duration of action, and therefore, these toxins can be used in lower doses (or in higher doses without any or other side effects) compared to known therapeutic clostridial toxins. One of the indicators of such advantageous properties (which is an increase in the therapeutic index) can be defined as the safety factor of the constructed clostridia toxin. From this point of view, the adverse effects of Clostridium toxin (caused by diffusion of the toxin from the injection site) can be experimentally assessed by measuring the percentage of body weight loss in a relevant animal model (for example, a mouse, where body weight loss is detected on the 7th day after administration ). In contrast, the desired effects of targeting a clostridial toxin can be experimentally assessed using a hindlimb digit abduction assessment (DAS) assay, a measure of the degree of muscle paralysis. The DAS assay can be performed by injecting 20 μl of clostridial toxin prepared in gelatin phosphate buffer into the gastrocnemius muscle/solar plexus of mice, followed by assessment of hindlimb digit abduction using the Aoki method (Aoki KR, Toxicon 39: 1815-1820; 2001). For the DAS assay, mice were briefly suspended by the tail in

- 5 046580 for the purpose of recording their characteristic start reflex, in which elongation of the hind limbs of mice and abduction of the fingers of the hind limbs are observed. After injection of clostridial toxin, various degrees of abduction of the hind digits are calculated on a five-point scale (0 = normal abduction - 4 = maximum reduction in abduction of the hind digits and lengthening of the foot).

The safety quotient of a clostridial toxin can then be expressed as the ratio of the amount of toxin required to produce a 10% reduction in body weight (measured at the maximum effect over the first seven days post-dose in mice) divided by the amount of toxin required to obtain a DAS score of 2. Therefore, It is desirable to obtain a high safety factor value, which indicates that a given toxin will effectively paralyze the target muscle with minimal unwanted side effects. The engineered toxin of the invention has a safety factor greater than that of the equivalent unmodified (native) botulinum toxin.

Thus, in one embodiment of the invention, the engineered clostridium toxin of the invention has a safety factor of at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), where the safety factor is calculated as the dose of toxin required to produce ~10% change in body weight (pg/mouse) divided by the DAS ED ₅₀ (pg/mouse) [ED ₅₀ = dose required to obtain a DAS score of 2].

In one embodiment, the engineered clostridium toxin of the invention has a safety factor of at least 10. In one embodiment, the engineered clostridium toxin of the invention has a safety factor of at least 15.

The constructed clostridium toxin according to the invention has at least one amino acid modification. At least one specified amino acid modification increases the pI of the clostridial toxin, as discussed above. In the context of the present invention, an amino acid modification is a modification of the amino acid sequence of a clostridial toxin. Such a modification can be made by replacing one amino acid in a sequence with another amino acid (ie, an amino acid substitution), by introducing a new amino acid into the sequence, or by deleting an amino acid in the sequence. Amino acids introduced into the amino acid sequence of a protein are also called amino acid residues.

The standard amino acids present in proteins are given below:

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table 2

Amino acid Side chain Aspartic acid Asp D Charged (acidic) Glutamic acid Glu E Charged (acidic) Arginine Arg R Charged (acidic) Lysine Lys TO Charged (main) Histidine His H Uncharged (polar) Asparagine Asn N Uncharged (polar) Glutamine Gin Q Uncharged (polar) Serin Ser S Uncharged (polar) Threonine Thr T Uncharged (polar) Tyrosine Tyr Y Uncharged (polar) Methionine Met M Uncharged (polar) Tryptophan Trp W Uncharged (polar) Cysteine Cys C Uncharged (polar) Alanin Ala A Uncharged (hydrophobic) Glycine Gly G Uncharged (hydrophobic) Valin Vai V Uncharged (hydrophobic) Leucine Leu L Uncharged (hydrophobic) Isoleucine He I Uncharged (hydrophobic) Proline Pro P Uncharged (hydrophobic) Phenylalanine Phe F Uncharged (hydrophobic)

The following amino acids are charged amino acids:

aspartic acid (negatively charged), glutamic acid (negatively charged), arginine (positively charged) and lysine (positively charged).

At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, and 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.

Uncharged polar amino acids (which can participate in the formation of hydrogen bonds) are the following amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan.

Uncharged hydrophobic amino acids are the following amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline and glycine.

Increasing the pI of the clostridia toxin can be achieved by introducing one or more amino acid modifications into the clostridia toxin to increase the ratio of positive charge to negative charge of the clostridia toxin.

In one embodiment of the invention, the at least one amino acid modification is selected from an amino acid substitution, an amino acid insertion, and an amino acid deletion.

In amino acid substitution, an amino acid residue that forms part of the amino acid sequence of the clostridium toxin is replaced with another amino acid residue. The substituting amino acid residue may be one of the 20 standard amino acid residues described above.

Alternatively, when performing an amino acid substitution, the replacement amino acid may be a non-standard amino acid (an amino acid that is not one of the 20 standard amino acids described above). Thus, for example, the replacement amino acid may be a basic non-standard amino acid, for example, L-ornithine, L-2-amino-3-guanidinopropionic acid or the D-isomers of lysine, arginine and ornithine. Methods for introducing non-standard amino acids into proteins are known to those skilled in the art and involve the synthesis of recombinant proteins using auxotrophic E. coli hosts in which the proteins are expressed.

When performing an amino acid insertion, an additional amino acid (an amino acid that is not normally present in the sequence) is introduced into the amino acid sequence of the clostridium toxin, which results in an increase in the total number of amino acid residues in the amino acid.

- 7 046580 given sequence. When performing an amino acid deletion, an amino acid residue is removed from the amino acid sequence of the clostridium toxin, which leads to a decrease in the total number of amino acid residues in the specified sequence.

Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known to those skilled in the art. For example, amino acid modifications can be introduced by modifying the DNA sequence encoding the clostridial toxin. This can be achieved by standard molecular cloning techniques, for example by site-directed mutagenesis, where short strands of DNA (oligonucleotides) encoding the desired amino acid(s) are introduced in place of the original coding sequence using the enzyme polymerase, or by insertion/deletion parts of a gene using various enzymes (for example, ligases and restriction endonucleases). Alternatively, the modified gene sequence may be chemically synthesized.

In one embodiment of the invention, at least one of the amino acid modifications is selected from replacing an acidic amino acid residue with a basic amino acid residue; replacing an acidic amino acid residue with an uncharged amino acid residue; replacing 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.

In a preferred embodiment of the invention, at least one of the amino acid modifications is a substitution that predominantly maintains the same number of amino acid residues in the clostridial toxin. In one embodiment of the invention, the substitution is selected from replacing an acidic amino acid residue with a basic amino acid residue; replacing an acidic amino acid residue with an uncharged amino acid residue; and replacing an uncharged amino acid residue with a basic amino acid residue. In one embodiment of the invention, the main amino acid residue is lysine or arginine. In one embodiment of the invention, the main amino acid residue is lysine. In one embodiment of the invention, the main amino acid residue is arginine. In one embodiment of the invention, if said replacement is the replacement of an acidic amino acid residue with an uncharged amino acid residue, then this means that the acidic amino acid residue is replaced with a corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine).

The constructed clostridial toxin according to the invention may have more than one amino acid modification. Thus, in one embodiment of the invention, the engineered clostridium toxin (described above) has from 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 of the invention, the engineered clostridium toxin (described above) has from 4 to 40 amino acid modifications. In one embodiment, the engineered clostridium toxin has at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid modifications. In one embodiment, the engineered clostridium toxin has at least 4 amino acid modifications (eg, at least 4 amino acid substitutions). Each of these amino acid modifications is an amino acid modification described above. Thus, each of these amino acid modifications increases the pI of the constructed clostridia toxin (compared to the pI of any other identical clostridia toxin that does not contain these amino acid modifications).

Any amino acid of the clostridial toxin (ie, amino acid residue) can be modified as described above, provided that such modification will result in an increase in the pI of the clostridial toxin (as described above). However, the present inventors have identified subseries of clostridial toxin amino acids that are particularly suitable targets for modification.

Preferred target amino acids may have certain valuable properties. Thus, for example, the preferred target amino acid may be: (i) an amino acid present on the surface; (ii) an amino acid located outside the secondary structure of the clostridia toxin protein; (iii) an amino acid located in the region of the clostridial toxin protein that does not play an important role in the function of the protein; (iv) an amino acid that is not conserved across different types, subtypes or serotypes of clostridial toxin; (v) an amino acid whose modification does not result in the formation of the predicted ubiquitination site; or (vi) any combination of the above amino acids.

As discussed above, clostridial toxins consist of two polypeptide chains, namely, a heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and a light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or Hc domain) and an N-terminal translocation component (HN domain).

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In one embodiment of the invention, at least one amino acid modification (described above) is present in the receptor-binding domain of the clostridium toxin (HC domain).

Examples of parent light chain sequences are: botulinum neurotoxin type A: amino acid residues 1-448, botulinum neurotoxin type B: amino acid residues 1-440, botulinum neurotoxin type C ₁ : amino acid residues 1-441, botulinum neurotoxin type D: amino acid residues 1- 445, botulinum neurotoxin type E: amino acid residues 1-422, botulinum neurotoxin type F: amino acid residues 1-439, botulinum neurotoxin type G: amino acid residues 1-441, tetanus neurotoxin: amino acid residues 1-457.

The above reference sequences should be considered as guide sequences since slight variations may be present in the sequences of different subserotypes. For example, US Application No. 2007/0166332 (which is incorporated herein by reference in its entirety) briefly describes various Clostridia sequences: botulinum neurotoxin type A: amino acid residues M1-K448, botulinum neurotoxin type B: amino acid residues M1-K441 , botulinum neurotoxin type C1: amino acid residues M1-K449, botulinum neurotoxin type D: amino acid residues M1-R445, botulinum neurotoxin type E: amino acid residues M1-R422, botulinum neurotoxin type F: amino acid residues M1-K439, botulinum neurotoxin type G: amino acid residues M1-K446, tetanus neurotoxin: amino acid residues M1-A457.

Examples of the original sequences of the HC domain of the clostridium toxin are: 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.

The Hc domain of a clostridium toxin (such as BoNT) has two characteristic structural features called the HCC and HCN domains. The amino acid residues involved in binding to the receptor are apparently localized mainly in the HCC domain.

In one embodiment of the invention, where at least one amino acid modification (described above) is present in the receptor-binding domain of the clostridium toxin ( _HC domain), the at least one amino acid modification is present in the HCN domain of the clostridium toxin (also called the H C domain stimulating translocation). In one embodiment, where at least one amino acid modification (described above) is present in the receptor binding domain of the clostridium toxin (HC domain), the at least one amino acid modification is present in the HCC domain of the clostridium toxin.

Examples of the original sequences of the HCN domain of clostridial toxin are: botulinum neurotoxin type A: amino acid residues 872-1110, botulinum neurotoxin type B: amino acid residues 859-1097, botulinum neurotoxin type C1: amino acid residues 867-1111, botulinum neurotoxin type D: amino acid residues 863-1098, botulinum neurotoxin type E: amino acid residues 846-1085, botulinum neurotoxin type F: amino acid residues 865-1105, botulinum neurotoxin type G: amino acid residues 864-1105, tetanus neurotoxin: amino acid residues 880-1127.

The positions of the above sequences may vary slightly depending on the serotype/subtype, and other examples of suitable clostridial toxin HCN domain sequences are:

botulinum neurotoxin type A: amino acid residues 874-1110, botulinum neurotoxin type B: amino acid residues 861-1097, botulinum neurotoxin type Ci: amino acid residues 869-1111, botulinum neurotoxin type D: amino acid residues 865-1098, botulinum neurotoxin type E: amino acids residues 848-1085, botulinum neurotoxin type F: amino acid residues 867-1105, botulinum neurotoxin type G: amino acid residues 866-1105,

- 9 046580 tetanus neurotoxin: amino acid residues 882-1127.

In one embodiment of the invention, the at least one amino acid modification (described above) is a modification of an amino acid residue located on the surface. Surface amino acid residues are present on the outside of the protein fold and are therefore accessible to the surrounding solvent compared to amino acid residues located inside the protein fold. The degree of accessibility of an amino acid residue on the surface, and therefore its accessibility to the surrounding solvent, depends on the position in the protein fold, as well as on the conformation that a given protein can take. Therefore, modification of an amino acid residue with a high degree of surface accessibility may have a greater effect on the isoelectric point of the protein than modification of an amino acid residue with a low degree of surface accessibility. Methods for determining the degree of accessibility of an amino acid residue on a surface are known to those skilled in the art. For example, to calculate the degree of accessibility of amino acid residues on the surface of a given protein, the ArealMol computer program (which is included in the CCP4 computer program package) can be used. Surface amino acid residues can also be identified by visual assessment of the protein crystal structure (eg, X-ray crystallography). In one embodiment, the surface amino acid residue has a total ArealMol value of at least 40.

In one embodiment of the invention, the at least one amino acid modification includes a modification of an amino acid residue selected from an aspartic acid residue, a glutamic acid residue, a histidine residue, a serine residue, a threonine residue, an asparagine residue, a glutamine residue, a cysteine residue, or a tyrosine residue. The present inventors have found that amino acid residues included in this group (negatively charged residues and polar residues) are particularly suitable targets for modification according to the invention. Without being limited to any particular theory, the authors only note that amino acid residues included in this group are present on the surface of the clostridia toxin more often than hydrophobic residues not included in this list.

In one embodiment, if the amino acid modification involves modification of an amino acid residue selected from aspartic acid, glutamic acid, histidine, serine, threonine, asparagine, glutamine, cysteine or tyrosine (described above), then such amino acid residue is replaced by lysine or arginine. Thus, in one embodiment of the invention, the negatively charged residue or polar residue is replaced by a positively charged residue, resulting in an increase in the ratio of positive charge to negative charge and an increase in the pI of the clostridial toxin.

In one embodiment, the at least one amino acid modification (described above) includes modification of an amino acid residue such as asparagine or glutamine (both of which are uncharged and polar). In one embodiment of the invention, an amino acid residue such as asparagine or glutamine is replaced with lysine or arginine (both of which are positively charged). In one embodiment of the invention, an amino acid residue such as asparagine or glutamine is replaced with lysine. In one embodiment of the invention, an amino acid residue such as asparagine or glutamine is replaced with arginine.

Residues such as asparagine and glutamine are particularly preferred for modification because they are polar, form only weak dipole bonds with other residues, and constitute 14% of the total molecule of a typical clostridial toxin (such as BoNT/A).

In one embodiment of the invention, the engineered clostridial toxin is BoNT/A. The original BoNT/A sequence has UniProtKB accession number P10845.

The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin BoNT/A.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, and GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase 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 value of any other identical BoNT/A that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one of the options

- 10 046580 implementation of the invention, the specified modification includes the replacement of an amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, and GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/A not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 8 8 6, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, and GLN 1229; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20), selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032 , ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, and ASP 1086; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30), selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1 229, ASN 1242, ASN 1243, SER 1274, and THR 1277; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase 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 value of any other identical BoNT/A that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30), selected from: ASN 8 8 6, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025 , ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277; and such amino acid modification(s) contribute(s) to increasing the isoelectric point (pI) of the engineered BoNT/A to a value that is at least

- 11 046580 at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT /A, not having the specified amino acid modification(s^). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30), selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1 229, ASN 1242, ASN 1243, SER 1274, and THR 1277; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 1 pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase 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 value of any other identical BoNT/A that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/A not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A 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 value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one of the options

- 12 046580 implementation of the invention, the specified modification includes the replacement of an amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0 .9 or 1) pI units higher than the pI value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A not having said amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase 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 value of any other identical BoNT/A that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/A not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5,

- 13 046580

0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0 .9 or 1) pI units higher than the pI value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A not having said amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase 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 value of any other identical BoNT/A that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/A not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5 or 6) selected from: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridium toxin is BoNT/A, then said engineered BoNT/A has a modification of the following seven

- 14 046580 amino acids: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052 or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A 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 value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0 .9 or 1) pI units higher than the pI value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of the following seven amino acids: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A not having said amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 6 or 7) , selected from: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase 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 value of any other identical BoNT/A that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 6 or 7) , selected from: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/A to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/A not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 6 or 7) , selected from: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine.

- 15 046580

In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of the following eight amino acids: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A 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 value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of the following eight amino acids: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (e.g., at least 0.5, 0.6, 0.7, 0.8, 0 .9 or 1) pI units higher than the pI value of any other identical BoNT/A that does not have the specified amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of the following eight amino acids: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and such amino acid modifications serve to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A not having said amino acid modifications. In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/A, then the engineered BoNT/A has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 6 or all 7 ), selected from: ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, HIS 1064, and GLN 1229; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11), selected from: ASN 886, ASN 905, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, HIS 1064, ASN 1080, ASN 1147 and GLN 1229; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/A, then said engineered BoNT/A has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all 13), selected from: ASN 886, ASN 905, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, HIS 1064,

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ASN 1080, ASN 1147, and GLN 1229; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI value of any other identical BoNT/A A, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, the engineered clostridial toxin is BoNT/B. The original BoNT/B sequence has the UniProtKB accession number P10844.

The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin BoNT/B.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/B, then the engineered BoNT/B has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252 and ASN 1273; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/B to increase 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 value of any other identical BoNT/B that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/B, then the engineered BoNT/B has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252 and ASN 1273; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/B to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/B not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/B, then the engineered BoNT/B has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252 and ASN 1273; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/B to a value that is at least one pI unit higher than the pI value of any other identical BoNT/B B, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, the engineered clostridial toxin is BoNT/C'1. The original BoNT/C'1 sequence has UniProtKB accession number P18640.

The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin BoNT/C1.

Thus, in one embodiment of the invention, if the constructed clostridial toxin is BoNT/C'1. then said engineered BoNT/C'1 has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP

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996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/B to increase 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 value of any other identical BoNT/C that does not have the specified ( s) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

Thus, in one embodiment of the invention, if the engineered clostridium toxin is BoNT/Ci, then the engineered BoNT/Ci has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15), selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079 and ASP 1081; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/C1 to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/C1 not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/C1, then said engineered BoNT/C1 has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) , selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079 and ASP 1081; and such amino acid modification(s) contribute(s) to increasing the isoelectric point (pI) of the engineered BoNT/C1 to a value that is at least one pI unit higher than the pI value of any other identical BoNT/C1 , not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, the engineered clostridial toxin is BoNT/D. The original BoNT/D sequence has the UniProtKB accession number P19321.

The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin BoNT/D.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/D, then the engineered BoNT/D has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20), selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033 , GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/D to increase 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 value of any other identical BoNT/D that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/D, then the engineered BoNT/D has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20), selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033 , GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/D to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/D not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one

- 18 046580 of the embodiments of the invention, said modification involves replacing an amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/D, then the engineered BoNT/D has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20), selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033 , GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/D to a value that is at least one pI unit higher than the pI value of any other identical BoNT/D D, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, the engineered clostridial toxin is BoNT/E. The original BoNT/E sequence has UniProtKB accession number Q00496.

The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin BoNT/E.

In one embodiment, if the engineered clostridial toxin is BoNT/E, then the engineered BoNT/E has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/E to increase 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 value of any other identical BoNT/E that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/E, then the engineered BoNT/E has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/E to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/E not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/E, then the engineered BoNT/E has a modification of at least one of the amino acids (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or 25), selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least one pI unit higher than the pI value of any other identical BoNT/E E, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, the engineered clostridial toxin is BoNT/F. The original BoNT/F sequence has the UniProtKB accession number YP 001390123.

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The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin BoNT/F.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/F, then the engineered BoNT/F has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20), selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997 , ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/F to increase 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 value of any other identical BoNT/F that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/F, then the engineered BoNT/F has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20), selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997 , ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/E to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/F not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is BoNT/F, then the engineered BoNT/F has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20), selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997 , ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/F to a value that is at least one pI unit higher than the pI value of any other identical BoNT/F F, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, the engineered clostridial toxin is BoNT/G. The original BoNT/G sequence has UniProtKB accession number Q60393.

The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin BoNT/G.

In one embodiment, if the engineered clostridial toxin is BoNT/G, then said engineered BoNT/G has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) , selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 1090; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/E to increase 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 value of any other identical BoNT/G that does not have the specified ) amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/G, then said engineered BoNT/G has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) , selected from: ASP 900, ASN

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909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075 and ASN 1090; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/E to increase to a value that is at least 0.5 (e.g., at least 0.5, 0 .6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical BoNT/G not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment, if the engineered clostridial toxin is BoNT/G, then said engineered BoNT/G has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) , selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 1090; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least one pI unit higher than the pI value of any other identical BoNT/E G, not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, the engineered clostridial toxin is TeNT. The original TeNT sequence has the UniProtKB accession number P04958.

The present inventors have identified certain amino acids that are preferred targets for amino acid modification in the clostridial toxin TeNT.

In one embodiment of the invention, if the engineered clostridial toxin is TeNT, then the engineered BoNT/G has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered BoNT/E to increase 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 value of any other identical TeNT that does not have the specified amino acid(s) modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is TeNT, then the engineered TeNT has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097; and such amino acid modification(s) cause the isoelectric point (pI) of the engineered TeNT to increase to a value that is at least 0.5 (e.g., at least 0.5, 0.6 , 0.7, 0.8, 0.9 or 1) pI units higher than the pI value of any other identical TeNT not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

In one embodiment of the invention, if the engineered clostridial toxin is TeNT, then the engineered TeNT has a modification of at least one of the amino acids (for example, at least 1, 2, 3, 4, 5, 10 or 15) selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097; and such amino acid modification(s) cause(s) to increase the isoelectric point (pI) of the engineered TeNT to a value that is at least one pI unit higher than the pI value of any other identical TeNT not having the specified amino acid modification(s). In one embodiment of the invention, the modification includes replacing the amino acid with lysine or arginine. In one embodiment of the invention, the modification includes replacing the amino acid with lysine. In one embodiment of the invention, the modification includes replacing the amino acid with arginine.

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Clostridia toxins from a wide range of different species can be used in the present invention. Thus, in the context of the present invention, the term clostridium toxin covers the toxins produced by the bacterium C. botulinum (botulinum neurotoxin serotypes A, B, C ₁ , 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 clostridial toxins or their derivatives derived from any of the above toxins. The term clostridium toxin also covers botulinum neurotoxin serotype H.

Botulinum neurotoxin (BoNT) is produced by the bacterium C. botulinum in the form of a large protein complex consisting of BoNT itself complexed with various accessory proteins. There are currently eight different classes of botulinum neurotoxins known, namely botulinum neurotoxins serotypes A, B, C1, D, E, F, G and H, and all of these toxins have similar structures and mechanisms of action. BoNTs of different serotypes can be identified by their inactivation by specific neutralizing antisera, and this serotype classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are also divided into different subtypes based on the percentage of amino acid sequence identity.

BoNTs are absorbed in the gastrointestinal tract, and after entering the general circulation, they bind to the presynaptic membrane of cholinergic nerve endings, which helps prevent the release of the neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F, and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/C ₁ , BoNT/A and BoNT/E cleave synaptosome-associated protein 25 kDa (SNAP-25), and BoNT/C1 cleaves syntaxin.

Tetanus toxin is produced by the bacterium C. tetani of one serotype. C. butyricum produces BoNT/E, and C. baratii produces BoNT/F.

The term clostridial toxin also includes modified clostridial toxins and derivatives thereof, including, but not limited to, the toxins described below. A modified clostridial toxin or derivative thereof may contain one or more amino acids that have been modified from those of the native (unmodified) form of the clostridial toxin, or they may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial toxin. For example, a modified clostridial toxin may have modified amino acid sequences in one or more domains compared to the sequences of the native (unmodified) clostridial toxin. Such modifications may alter the functional properties of the toxin, such as its biological activity or persistence. Thus, in one embodiment of the invention, the engineered clostridia toxin of the invention is an engineered modified clostridia toxin or an engineered modified derivative of clostridia toxin or an engineered derivative of clostridia toxin.

The modified clostridial toxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified HC domain), wherein the modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial toxin. Such modifications in the _HC domain may include modification of residues in the ganglioside-binding site of the HC domain or in the protein binding site (SV2 or synaptotagmin), where this modification leads to a change in the level of binding to the ganglioside receptor and/or to the protein receptor of nerve cells - targets. Examples of such modified clostridial toxins are described in WO 2006/027207 and WO 2006/114308, which are incorporated herein by reference in their entirety.

The modified clostridium toxin may have one or more modifications in the amino acid sequence of the light chain, for example, modifications in the substrate binding domain or in the catalytic domain, where these modifications can alter or modify the specificity of the modified LC for the SNARE protein. Examples of such modified clostridial toxins are described in WO 2010/120766 and US Application 2011/0318385, which are incorporated herein by reference in their entirety.

The modified clostridial toxin may have one or more modifications that increase or decrease the biological activity and/or biological persistence of the modified clostridial toxin. For example, a modified clostridial toxin may contain a leucine- or tyrosine-based motif, wherein the motif promotes an increase or decrease in the biological activity and/or biological persistence of the modified clostridial toxin. Suitable leucine-based motifs are xDxxxLL, xExxxLL, xExxxIL and xExxxLM (where x is any amino acid). A suitable tyrosine-based motif is Y-x-x-Hy (where Hy is a hydrophobic amino acid). Examples of modified clostridial toxins containing leucine and tyrosine motifs are described in WO 2002/08268, which is incorporated herein by reference in its entirety.

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The term clostridial toxin covers hybrid and chimeric clostridial toxins. The hybrid clostridial toxin contains at least a portion of a light chain derived from one clostridial toxin or a subtype thereof and at least a portion of a heavy chain derived from another clostridial toxin or a subtype thereof. In one embodiment, the hybrid clostridial toxin may comprise a full-length light chain derived from one subtype of clostridial toxin and a heavy chain derived from a different subtype of clostridial toxin. In another embodiment of the invention, the chimeric clostridial toxin may comprise a portion (eg, a binding domain) of a heavy chain of a clostridial toxin of one subtype and a portion of a heavy chain derived from a clostridial toxin of another subtype. Likewise or alternatively, the therapeutic element may comprise light chain portions derived from various clostridial toxins. Such hybrid or chimeric clostridia toxins can be used, for example, as vehicles for delivering the therapeutically valuable elements of these clostridia toxins to patients with immunological resistance to a given subtype of clostridia toxin; patients in whom the concentration of receptors for the heavy chain binding domain of a given clostridial toxin may be below average; or patients who may have a protease-resistant variant of a membrane or vesicular toxin substrate (eg, SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial toxins are described in US Application Publication 8,071,110, which is incorporated herein by reference in its entirety. Thus, in one embodiment of the invention, the engineered clostridial toxin of the invention is an engineered hybrid clostridium toxin or an engineered chimeric clostridium toxin.

The term clostridial toxin covers retargeted clostridial toxins. Retargeted clostridial toxin is a clostridial toxin that has been modified to include an exogenous ligand known as a targeting molecule (TM). The TM is selected so that it provides binding specificity to the desired target cell, and during the retargeting process, the native binding portion of the clostridia toxin (eg, the HC domain or HCC domain) can be removed. Retargeting technology is described, for example, in EP-B-0689459; WO 1994/021300; EP-V-0939818; US 6461617; US 7192596; WO 1998/007864; EP-V-0826051; US 5989545; US 6395513; US 6962703; WO 1996/033273; EP-B-0996468; US 7052702; WO 1999/017806; EP-B-1107794; US 6632440; WO 2000/010598; WO 2001/21213; WO 2006/059093; WO 2000/62814; WO 2000/04926; WO 1993/15766; WO 2000/61192; and WO 1999/58571, all of which are incorporated herein by reference in their entirety. Thus, in one embodiment of the invention, the engineered clostridia toxin of the invention is an engineered retargeted clostridia toxin.

The present invention also relates to clostridial toxins that have an HC-native protease cleavage site. In such clostridial toxins, the native protease cleavage site (also known as the activation site described above) has been modified or replaced by a protease cleavage site that is not native to the clostridial toxin (ie, an exogenous cleavage site). Cleavage at this site requires an exogenous protease, which allows for better regulation of the duration and location of cleavage events. HC-native protease cleavage sites that can be used in clostridial toxins are:

enterokinase (DDDDKj), factor Xa (IEGRj/IDGRj),

TEV (tobacco etch virus) (ENLYFQIG), thrombin (LVPRjGS),

PreScission (LEVLFOJGP).

Other protease cleavage sites are recognition sequences that are cleaved by a noncytotoxic protease, such as the light chain of a clostridial neurotoxin. Such sites are sequences that recognize SNARE proteins (eg, SNAP-25, syntaxin, VAMP), which are cleaved by noncytotoxic proteases such as the clostridial neurotoxin light chain. Clostridial toxins containing HC-native protease cleavage sites are described in US Pat. No. 7,132,259, EP1206554-B2, and US Application No. 2007/0166332, which are incorporated herein by reference in their entirety. The term protease cleavage site also covers an intein, which is a self-cleaving sequence. The autosplicing reaction is regulated, for example, by changing the concentration of the reducing agent present.

The present invention also covers clostridial toxins containing a destructive cleavage site. In these clostridial toxins, the HC native protease cleavage site is introduced into the clostridial toxin at a position selected such that cleavage at the site results in decreased activity or inactivation of the clostridial toxin. The destructive protease cleavage site may be susceptible to local protease cleavage such that the clostridial toxin, once administered, may migrate to a non-target position. Suitable

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The HC native protease cleavage sites are those described above. Clostridial toxins containing a destructive cleavage site are described in WO 2010/094905 and WO 2002/044199, which are incorporated herein by reference in their entirety.

The engineered clostridial toxins of the invention, especially the light chain component thereof, can be PEGylated to improve stability, eg, increase the duration of action of the light chain component. PEGylation is particularly preferred if the light chain contains a BoNT/A, B or C ₁ protease. PEGylation preferably involves adding PEG to the N-terminus of a component such as a light chain. For example, the N-terminus of the light chain may be extended by the addition of one or more amino acid residues (eg, cysteine), which may be the same or different. One or more of these amino acid residues may have its own PEG molecule attached to these residues (eg, a covalently linked molecule). An example of such technology is described in WO 2007/104567, which is incorporated herein by reference in its entirety.

The engineered clostridia toxins of the invention may not contain complexing proteins that are typically present in the natural clostridia toxin complex.

The engineered clostridium toxin according to the invention may also contain a limited number of non-standard amino acids. Thus, in the constructed clostridial toxins according to the invention, amino acids can be replaced not only by 20 standard amino acids, but also by non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine). Amino acid residues of the clostridia polypeptide can be replaced by a limited number of non-conservative amino acids; amino acids that are not encoded by the genetic code; and unnatural amino acids. The engineered clostridial toxins of the invention may also contain unnatural amino acid residues.

Unnatural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4fluorophenylalanine.

Specialists know several methods for introducing unnatural amino acid residues into proteins. For example, an in vitro system can be used in which nonsense mutations have been inhibited by chemically aminoacylated suppressor tRNAs. Methods for the synthesis of amino acids and methods for aminoacylation of tRNA are known to specialists. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system containing E. coli S30 extract and commercially available enzymes, as well as other reagents. Proteins are purified using 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 carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired unnatural amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine).

An unnatural amino acid is introduced into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33:7470-6, 1994.

The engineered clostridial toxins of the invention can be produced using recombinant nucleic acid techniques. Thus, in one embodiment of the invention, the engineered clostridium toxin (described above) is a recombinantly engineered clostridium toxin.

In another aspect, the present invention provides a nucleic acid (eg, DNA) containing a nucleic acid sequence encoding the engineered clostridium toxin described above. In one embodiment of the invention, the nucleic acid sequence is obtained as part of a DNA vector containing a promoter and terminator.

In a preferred embodiment of the invention, the vector has a promoter selected from promoters induced by the agent under typical induction conditions:

Tas (hybrid), IPTG, 0.2 mM (0.05-2.0 mM),

AraBAD, L-arabinose, 0.2% (0.002-0.4%),

T7-lac operator, IPTG, 0.2 mM (0.05-2.0 mM).

In another preferred embodiment of the invention, the vector has a promoter selected from promoters induced by the agent under typical induction conditions:

Tas (hybrid), IPTG, 0.2 mM (0.05-2.0 mM),

AraBAD, L-arabinose, 0.2% (0.002-0.4%),

T7-lac operator, IPTG, 0.2 mM (0.05-2.0 mM),

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T5-1ac operator, IPTG, 0.2 mM (0.05-2.0 mM).

The nucleic acid molecules according to the invention can be obtained by any suitable method known to those skilled in the art. Thus, nucleic acid molecules can be obtained by chemical synthesis methods. Alternatively, the nucleic acid molecules of the invention can be obtained by molecular biology methods.

The DNA construct of the invention is preferably designed in silico and then synthesized using standard DNA synthesis methods.

The data obtained for the above nucleic acid sequence is optionally modified by codon shifting according to the end host cell (eg E. coli) expression system used.

In one embodiment, the nucleic acid sequence encoding the engineered clostridium toxin described above is a nucleic acid sequence that is at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98 or 99%) identical to the nucleic acid sequence selected from SEQ ID NN: 3, 5, 7 and 9.

In one aspect, the present invention provides a nucleic acid sequence that is at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, or 99%) identical to a nucleic acid sequence selected from SEQ ID NN: 3, 5, 7 and 9. In one embodiment, the nucleic acid sequence is at least 90% identical to the nucleic acid sequence selected from SEQ ID NN: 3, 5, 7 and 9.

The present invention also relates to polypeptides encoded by the nucleic acid sequences described above. Thus, in one aspect, the present invention provides a polypeptide comprising an amino acid sequence that is at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, or 99%) identical to the amino acid sequence selected from SEQ ID NN: 4, 6, 8 and 10. In one embodiment, the amino acid sequence is at least 90% identical to the amino acid sequence selected from SEQ ID NN: 4, 6, 8 and 10 .

In one embodiment, the engineered clostridial toxin of the invention is the engineered BoNT/A described above, wherein said engineered BoNT/A comprises an amino acid sequence that is at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) identical to the amino acid sequence selected from SEQ ID NN: 4, 6, 8 and 10.

In one embodiment, the engineered clostridial toxin of the invention is the engineered BoNT/A described above, wherein the engineered BoNT/A comprises (or consists of) the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10.

In one aspect, the present invention provides a polypeptide that includes (or consists of) the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10.

In one aspect, the present invention provides a nucleic acid encoding an engineered clostridium toxin described above, wherein said nucleic acid comprises a nucleic acid sequence that is at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) identical to a nucleic acid sequence selected from SEQ ID NN: 3, 5, 7, and 9. In one embodiment, the nucleic acid comprises a nucleic acid sequence acid (or consists of it) SEQ ID NO: 3, 5, 7 or 9.

In one aspect, the present invention provides a nucleic acid comprising (or consisting of) a nucleic acid sequence of SEQ ID NO: 3, 5, 7 or 9.

The percentage of sequence identity between two or more nucleic acid or amino acid sequences depends on the number of identical positions in those sequences. For example, % identity can be calculated as the ratio of the number of identical nucleotides/amino acids to the total number of nucleotides/amino acids multiplied by 100. Percent sequence identity can also be calculated taking into account the number of gaps and the total length of each gap that must be introduced to optimize the alignment two or more sequences. Comparing sequences and determining the percent identity of two or more sequences can be accomplished using specially designed mathematical algorithms well known to those skilled in the art, such as BLAST.

In one aspect, the present invention relates to a method for producing a single-chain engineered clostridial protein toxin having a light chain and a heavy chain, the method comprising expressing a nucleic acid (the nucleic acid described above) in a suitable host cell; lysing the host cell to produce a host cell homogenate containing the single-chain engineered protein clostridium toxin; and isolating said single-chain engineered clostridial protein toxin.

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In another aspect, the present invention relates to a method for activating an engineered clostridia toxin, wherein the method comprises producing a single chain engineered protein clostridia toxin as described above; contacting the 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 a disulfide bond.

The engineered clostridial toxins of the invention can be used to prevent or treat certain diseases and conditions, as well as for cosmetic purposes. Thus, in another aspect, the present invention relates to the use of the engineered clostridial toxin described above in medicine.

In a related aspect, the present invention relates to the use of the engineered clostridial toxin described above for the purpose of preventing or treating a disease or condition selected from: strabismus; blepharospasm;

heterotropia; dystonia (eg, spastic dystonia, mandibular dystonia, focal dystonia, tardive dystonia, oropharyngeal dystonia, limb dystonia, cervical dystonia); torticollis (eg, spasmodic torticollis);

reduction of cellular/muscular activity (through inhibition or inactivation of SNAREs), which can be prevented by methods used in cosmetology (using cosmetics); neuromuscular disease or impaired motility of the eyeball (for example, concomitant strabismus, vertical strabismus, paralysis of the lateral rectum, nystagmus, disthyroid myopathy);

writing cramp;

blepharospasm; bruxism; Wilson's disease; tremor; tics; segmental dancing eye syndrome; spasms; spasticity caused by chronic multiple sclerosis; spasticity leading to bladder dysfunction; animus; spasms in the back; calf muscle cramps (rider's disease); tension headache; levator pelvis syndrome; spina bifida syndrome; tardive dyskinesia; Parkinson's disease; stiffness; spasms in the hemifacial area; eyelid lesions; cerebral palsy; focal spasticity; spastic colitis; neurogenic bladder; anism; spasticity of the limbs; tics; tremors; bruxism; cracks in the anal area; achalasia; dysphagia; tearfulness; hyperhidrosis;

excessive salivation; excessive secretion of gastric juice; muscle pain (such as pain caused by muscle spasms); headache (eg tension headache); deep wrinkles in the eyebrow area; cancer; diseases of the uterus; genitourinary tract disorders; genitourinary-nervous disorders; chronic neurogenic inflammation and smooth muscle disorders.

The present invention uses a pharmaceutical composition containing an engineered clostridium toxin in combination with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or a pharmaceutically acceptable salt.

The engineered clostridial toxins of the invention can be administered orally, parenterally, by continuous infusion, by inhalation or by topical application. Injectable compositions may be administered in the form of solutions, suspensions or emulsions, or in the form of dry powders which, prior to use, are dissolved or suspended in a suitable vehicle.

In the case of topical administration of an engineered clostridial toxin, the engineered clostridial toxin may be formulated as a cream (eg, for topical application) or as a subdermal injection.

Methods of local delivery may include the use of an aerosol or other spray (eg, administration through an inhaler). Accordingly, an aerosol composition comprising an engineered clostridial toxin provides delivery of the toxin to the lungs and/or other nasal and/or bronchial or respiratory tracts.

The constructed clostridial toxins according to the invention can be administered to the patient by intrathecal or epidural injection into the spinal column at the level of the spinal segment responsible for the innervation of the affected organ.

The preferred route of administration is laparoscopy and/or local, and in particular intramuscular injection.

The engineered clostridial toxins of the invention are administered in doses sufficient to achieve the desired therapeutic effect. It should be noted that the required dosage range depends on the specific nature of the engineered clostridial toxin or its composition; on the method of administration and properties of the composition; on the age of the patient; on the nature, extent or severity of the patient's condition; from contraindications, if any, and from the prescription of the attending physician. These doses may vary and can be adjusted using standard empirical routine optimization techniques.

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Suitable daily doses (per kg of patient body weight) are in the range of 0.0001-1 ng/kg, preferably 0.0001-0.5 ng/kg, more preferably 0.002-0.5 ng/kg, and particularly preferably 0.004- 0.5 ng/kg. The unit dose may vary from less than 1 picogram to 30 ng, and is typically in the range of 0.01 to 1 ng, and such dose may be administered daily or, preferably, less frequently, such as once a week or every six months. months.

A particularly preferred dosing schedule is to administer 0.05 ng of engineered clostridial toxin in a 1x dose. In this case, preferred doses are in the range of 1x - 100x (ie, 0.05-5 ng).

Liquid dosage forms typically contain an engineered clostridium toxin and a pyrogen-free sterile carrier. The engineered clostridium toxin, depending on the vehicle used and its concentration, may be dissolved or suspended in the vehicle. When preparing solutions, the engineered clostridium toxin can be dissolved in a vehicle and then the resulting solution can be made isotonic, if necessary, by adding sodium chloride followed by sterilization by filtration through a sterile filter under aseptic conditions, after which the resulting solution is filled with suitable sterile containers or ampoules and these containers or ampoules are hermetically sealed. Alternatively, if the stability of the solution is acceptable, the solution, contained in hermetically sealed containers, can be sterilized by autoclaving. Preferably, additives such as buffering agents, solubilizers, stabilizers, preservatives or bactericidal, suspending or emulsifying agents and/or local anesthetics may be dissolved in the carrier.

Dry powders, which are dissolved or suspended in a suitable vehicle before use, can be prepared by filling a sterile container with pre-sterilized ingredients using sterile sterilization techniques. Alternatively, these ingredients may be dissolved in suitable containers using sterilization techniques under sterile conditions. The product is then freeze-dried and the containers are hermetically sealed under aseptic conditions.

Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in essentially the same manner, except that the sterile components, instead of being dissolved, are suspended in a sterile vehicle, in which case sterilization cannot be done by filtering. These components can be isolated in a sterile state, or, alternatively, they can be sterilized after their isolation, for example by gamma irradiation.

To ensure uniform distribution of the components, a suspending agent, for example polyvinylpyrrolidone, is preferably included in the composition(s).

In accordance with the present invention, administration can be accomplished using various delivery methods, including microparticle encapsulation, delivery using a viral system, or high pressure aerosol nebulization.

Description of drawings

Fig. 1. Isoelectric focusing (IEF) in a gel containing cationic constructs.

Fig. 2. Percentage of SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) under the influence of Cat5v2(K1064H/N954K) (A), Cat5v2(K1064H/N886K) (B) and Cat5v2(K1064H/N1025K) (C), and sum dose pEC ₅₀ for nBoNT/A1. (A, B, C) Fetal rat spinal cord neurons were cultured for three weeks, treated with Cat5v4 for 24 h, and then Western blotted using a SNAP-25-specific antibody. Data are presented as mean ± avg. sq. osh. for three independent experiments performed in triplicate. (D) Relative activities of Cat5v2(K1064H/N886K), Cat5v2(K1064H/N954K), and Cat5v2(K1064H/N1025K) toward nBoNT/A1 (List Biological Laboratories) in a SNAP-25 cleavage activity assay in rat eSCN. Each point corresponds to an individual batch and represents the average of 3 independent determinations of pEC ₅₀ using a concentration-response curve (CRC) constructed from 8 points. Each CRC concentration was assessed in triplicate. Activity comparison data are presented as the average of the parties included in the list, cumulative data, n=24. Data are presented as mean ± avg. sq. osh. for n=3 games on Cat5v4.

Fig. 3. Activity (t ₅₀ ) of nBoNT/A1 and Cat5v4 in an assay performed on the right or left dome of the mouse phrenic nerve (mPNHD). Right or left murine phrenic nerve dome tissue was incubated with Cat5v4 or native BoNT/A1 as indicated in the figure. Diaphragm contractility was recorded until contractions were no longer detected or after 140 min. Each point corresponds to independent definitions. The value of t ₅₀ means the time required to inhibit the contractility of the right or left dome of the mouse diaphragm by 50%.

Sequences SEQ ID NO: 1.

BoNT/A1 nucleic acid sequence. SEQ ID NO: 7.

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Designed BoNT/Al Cat-C nucleic acid sequence. SEQ ID NO: 8.

Designed amino acid sequence of BoNT/A1 Cat-C. SEQ ID NO: 9.

Designed BoNT/A1 Cat-D nucleic acid sequence.

SEQ ID NO: 10. Designed amino acid sequence of BoNT/A1 Cat-D.

Examples

The following examples are provided for the purpose of illustrating specific embodiments of the invention and should not be construed as limiting the scope of the invention as claimed.

Example 1.

Three different variants of the engineered BoNT/A1 molecule were prepared in accordance with the present invention.

The amino acids to be modified (at mutation sites) were selected based on a number of different criteria.

Residues were replaced according to the following criteria:

1) type of remainder;

2) the degree of accessibility of the residue on the surface;

3) localization in the secondary/tertiary structure;

4) localization of known functional domains of BoNT;

5) the degree of sequence conservation for the BoNT/A or BoNT/E subtypes;

6) the possibility of introducing an additional ubiquitination site.

In this example, asparagine (Asn, N) and glutamine (Gln, Q) were chosen for mutation because they are polar, have a similar size to Lys, form only weak dipole bonds with other residues, and make up 14% of all residues in a molecule.

The Asn and Gln residues, which are visually detectable on the surface of the molecule, were identified from the crystal structure of BoNT/A1 (PDB ID: 3BTA). This means that all replaced residues have an external charge. From this list, you can exclude those residues that are least suitable for replacement in accordance with paragraphs 3-5 of the above selection criteria (re-iteration operation).

Non-conserved residues in BoNT/A1 were identified through their alignment with residues in BoNT/A of other subtypes and functionally similar serotypes of BoNT/E. Residues identified as core residues in other sequences were selected as the best candidates for replacement.

After conducting successive rounds of iteration according to the selection criteria specified in the sections above, a final list of candidate residues was identified. These residues were screened for the possible presence of additional ubiquitination consensus sequences (using the CKSAAP_UbSite server). Several identified lysine residues were removed by replacing them with arginine.

The final representative synthesized cationic constructs having BoNT/A1 sequences are listed below and are designated Cat-A, Cat-B and Cat-C. Each construct has a molecular weight of 149637 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.

Example 2.

The amino acid sequences BoNT/B, F and E were evaluated for candidate residues that could be replaced by Lys or Arg residues. This preliminary assessment allows the identification of residues that can be replaced to produce a BoNT/B, E or F protein having a higher pI

The primary sequence of BoNT/B (Ac: P10844), BoNT/E (Ac: Q00496) and BoNT/F (Ac: P30996) was analyzed, and the amino acid composition of this sequence is systematized in the following table:

Table 3

Serotype Theoretical pl Net charge at pH 7.4 No. Asn and Gln No. Asp and Glu BoNT/B 5, 3 -23 179 156 BoNT/E 6, 2 -7 160 132 BoNT/F 5, 4 -22 169 161

As can be seen from the table, the indicated amino acid sequences have the same large number of polar Asn/Gln residues as observed in BoNT/A1. These sequences also contain a relatively large number of acidic (Asp/Glu) residues that can be replaced

- 28 046580 either corresponding neutral residues (Asn/Gln) or basic residues (Lys or Arg).

Example 3.

Identification of amino acids preferred for modification of clostridia toxin.

For the sequences BoNT/A, BoNT/B and BoNT/E, data on their full-length structure is available, however, for the remaining four serotypes, based on information about the sequences and their structural homology, a theoretical model was built using the computer program LOOPP.

Each structure was analyzed using the ArealMol program (part of the CCP4 software package), and the available residues were identified as residues having a total number of more than 40. From this list, residues with polar side chains were selected, and preference was given to residues that are either acidic (Asp and Glu) or have an acceptor side chain with an H bond (Asn and Gln). The final stage of the computer analysis involves selecting residues located between the α-helices and β-strands, according to the analysis carried out on the Stride server. The structure of each molecule was visually assessed to identify residues located in border regions, and these residues were discarded.

For BoNT/A1, the list of preferred residues was supplemented with functionally non-conserved residues representing at least 90% of all residues of the matched sequences [residues with large non-polar side chains (Met, Pro, Phe, Trp) were considered equivalent; residues with small nonpolar side chains (Gly, Ala, Val, Leu, Ile) were considered equivalent; residues with acidic side chains (Asp, Glu) were considered equivalent; and residues with basic side chains (Arg, Lys) were considered equivalent]. More specifically, those non-conserved residues that were identified as core residues constituting at least 10% of the total residues in the sequences and the non-conserved Asn, Gln, Asp or Glu residues present in the original sequence were selected as candidates.

For the remaining serotypes, multiple sequence alignments of different subtypes were performed to identify functionally non-conserved residues, which were identified as core residues representing at least 10% of the total residues in the sequences.

Below are the preferred clostridium toxin amino acids for modification:

BoNT/A:

used sequences:

identification numbers:

BoNT/A: P10845,

BoNT/B: P10844,

BoNT/Ci Р18640,

BoNT/D: P19321,

BoNT/E: Q00496,

BoNT/F: YP_001390123,

BoNT/G: Q60393,

TeNT: P04958.

Source of structural data.

The crystal structures of BoNT/A (3BTA.pdb), BoNT/B (1EPW) and BoNT/E (3FFZ.pdb) were obtained from RCSB.

Homology modeling of BoNT/C.'|. BoNT/D, BoNT/F, BoNT/G and TeNT were carried out using the LOOPP program and the following sequences, respectively: P18640, P19321, YP_001390123, Q60393 and P04958.

Structural analysis.

Available residues were determined using the ArealMol program included in the CCP4 software package.

Secondary structure assignment was performed using the Stride program.

Borderline residues were determined by visual assessment using the RasMol program. Sequence analysis.

Full-length BoNT sequences were obtained from NCBI.

Alignment was performed using the ClustalX computer program.

Example 4.

Cloning, expression and purification.

DNA constructs encoding the engineered BoNT/A molecules described in Example 1 were synthesized, cloned into the pJ401 expression vector, and then transferred into E. coli BL21 (DE3). As a result, overexpression of soluble recombinant proteins Cat-A, Cat-B and Cat-C in BL21(DE3) E. coli was achieved.

Recombinantly engineered BoNTs were purified from E. coli lysates using classical methods

- 29 046580 chromatography. First, a purification step using a cation exchange resin was carried out, and then an intermediate purification step was carried out using a hydrophobic resin. Thereafter, the recombinant single-chain engineered BoNT was cleaved through proteolysis to obtain activated double-chain BoNT. A final purification step was then carried out to remove remaining impurities.

Example 5.

Characterization of purified engineered BoNTs.

The constructed BoNTs described in Example 1 above were characterized experimentally in the manner described below.

The pI measurement showed that the engineered BoNTs had a higher isoelectric point value than the unmodified (native) BoNT/A1 - see FIG. 1 and the table below.

Table 4

BoNT/Al molecule pl (calculated) pl (observable) Constructed "Cat-A" [Cat5v2(K1064H/N886K)] 6, 9 ~8.0 Constructed "Cat-B" [Cat5v2(K1064H/N954K)] 6, 9 ~8.0 Constructed "Cat-C" [Cat5v2(К1064H/N1025К)] 6, 9 7.8-8.0 Native BoNT/Al [nBoNT/Al] 6, 05 ~7.4

The ability of engineered BoNTs to enter neurons and cleave SNAP-25 (a target of BoNT/A1) was assessed using fetal rat spinal cord neurons (eSCN). In fig. Figure 2 shows that the engineered BoNT retained the same ability to enter the neuron and cleave SNAP-25 as native BoNT/A1.

The activity of the engineered BoNTs was further assessed using an assay performed using the right or left mouse phrenic nerve dome (mPNHD). In fig. Figure 3 shows that the engineered BoNTs retained the same ability to inhibit contraction of the right or left dome of the mouse diaphragm as native BoNT/A1.

An in vivo Digit Abduction Score (DAS) assay was performed to determine the activity as well as the safety factor of the engineered BoNT compared to the activity and safety factor of native BoNT/A1. Both of these molecules had a higher safety factor than native BoNT/A1 and were slightly more active. The obtained data are presented below (Table 4).

Table 4

Molecule DAS ED ₅ o (pg/mouse) Dose DAS 4 (pg/mouse) Dose for -10% ABW (pg/mouse) Safety factor Native BoNT/Al (n=5) 2 10-20 9.9-14.5 7 Constructed "Cat-A" 1.16 10-20 27.4 24 Constructed "Cat-B" 1.79 25 47, 6 27

DAS ED ₅₀ : Calculated dose to induce DAS 2.

DAS 4 Dose: Experimental DAS 4 inducing dose.

BW: Body weight.

Dose for ~10% ABW: Calculated dose to cause a 10% reduction in BW compared to BW on day 0.

Safety quotient: Dose for ~10% ABW/DAS ED50 The safety quotient is a measure of the post-BoNT adverse effect (weight loss) relative to BoNT activity (half-maximal hindlimb digit abduction score (DAS)). This ratio was calculated as the ratio of ~10% body weight (BW) to DAS ED50, where ~10% BW means the amount

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Claims (4)

The amount of BoNT (pg/animal) required to achieve a 10% reduction in body weight, and ED ₅₀ means the amount of BoNT (pg/animal) sufficient to achieve DAS=2. The DAS assay was performed by injecting 20 μl of engineered clostridial toxin prepared in gelatin phosphate buffer into the gastrocnemius muscle/solar plexus of a mouse, followed by assessment of hindlimb digit abduction as described previously in Aoki (Aoki KR, Toxicon 39: 1815- 1820; 2001). Example 6 Another engineered clostridial toxin according to the invention was prepared in accordance with the criteria specified above in Example 1. This cationic construct was also derived from BoNT/A1 and had a calculated pI of 7.4 and a molecular weight of 149859. This construct was designated Cat-D. Constructs Cat-A, Cat-B, and Cat-C contained residues replaced by lysine, and Cat-D contained residues replaced by arginine. Cat-D: N1188R, D1213R, G1215R, N1216R, N1242R, N1243R, S1274R, T1277R. Example 7. Treatment of a patient suffering from cervical dystonia. A 50-year-old woman with spasmodic torticollis was an inpatient at the clinic where she was treated by injecting a therapeutically effective amount of standard BoNT/A into the neck muscle, however, this patient experienced dysphagia caused by spread of the toxin into the oropharynx. This patient was injected into the neck muscles with approximately 1.5 ng (or more) of engineered BoNT/A according to the invention. After 3 to 7 days, the patient's degree of torticollis was significantly reduced without any dysphagia, and the patient was able to keep her head and shoulders in a normal position for at least five months. Since the engineered BoNT/A molecule was retained in the tissue for a longer period of time and had little penetration into other organs, the attending physician could prescribe more drug product without any fear of side effects, and this increase in dose would provide more long-term effect of the drug. Example 8. Treatment of a patient suffering from blepharospasm. A 47-year-old man suffering from blepharospasm was inpatient treatment at the clinic. This patient was treated by injecting 5 pg - 25 pg of engineered BoNT/A according to the invention into the lateral tarsal circle muscle of the upper eyelid and into the lateral tarsal circle muscle of the lower eyelid. After about a week, this patient's symptoms subsided, and after at least five months, the symptoms of the disease completely disappeared, and no blepharoptosis was observed. The high level of safety of the polypeptide according to the invention allowed the attending physician to increase the dose of this polypeptide and thereby increase the duration of its clinical effect. Example 9. A 27-year-old man suffering from cerebral palsy, which included symptoms such as a deformed equine foot and difficulty walking, was an inpatient at the clinic. This patient was treated with the therapeutically effective BoNT/A, which resulted in an improvement in his gait, but this improvement was accompanied by muscle weakness and pain in the extremities. This patient was treated by injecting approximately 20 pg/kg of the engineered BoNT/A of the invention into each of two sites on the medial and lateral dome of the gastrocnemius muscle of the affected lower limb(s). After a week, the patient's gait improved without any obvious side effects, and after at least four months, the symptoms of this disease completely disappeared. The possibility of increasing the dose of this drug made it possible to continue treatment and thereby increase the duration of its action. CLAIM 1. A constructed therapeutic clostridium neurotoxin containing at least two amino acid modifications, where at least two of said amino acid modifications increase the isoelectric point (pI) of the constructed therapeutic neurotoxin to a value that is at least 0.2 units higher than the pI of another identical a clostridial neurotoxin not containing at least two of these amino acid modifications, wherein the at least two amino acid modifications are located in a clostridial toxin receptor binding domain (HC domain), wherein the engineered therapeutic clostridial neurotoxin is botulinum neurotoxin A (BoNT/A) having a pI of at least 6.6, wherein the engineered therapeutic neurotoxin contains a modification of at least - 31 046580 two amino acids selected from: acidic amino acid residues: GLU 920, GLU 992, ASP 1058, GLU 1081, GLU 1083 and ASP 1086 and/or uncharged amino acid residues: ASN 886, ASN 905, GLN 915, ASN 918, ASN 930, ASN 954, SER 955, GLN 991, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, HIS 1064, ASN 1080 and GLN 1229, where at least two of these amino acid modifications contain: (i) replacing an acidic amino acid residue with a lysine residue or an arginine residue; (ii) replacing an acidic amino acid residue with a histidine residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, a tyrosine residue, a methionine residue, a tryptophan residue, a cysteine residue, an alanine residue, a glycine residue, a valine residue, a leucine residue, an isoleucine residue, a proline or phenylalanine residue; (iii) replacing an uncharged amino acid residue with a lysine residue or an arginine residue; (iv) insertion of a lysine residue or an arginine residue; or (v) deletion of an acidic amino acid residue, and wherein the engineered therapeutic clostridial neurotoxin is a double-chain engineered therapeutic clostridial neurotoxin, wherein a light chain and a heavy chain are linked to each other by a disulfide bond, prepared by a method comprising providing a single-chain engineered therapeutic clostridial neurotoxin containing a polypeptide sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 4, 6 and 8, or a single-chain engineered therapeutic clostridial neurotoxin encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 3, 5, and 7, and contacting the single-chain engineered therapeutic clostridial neurotoxin with a protease that cleaves the polypeptide at a cleavage site located between the light chain and the heavy chain, thereby converting the single-chain engineered therapeutic clostridial neurotoxin into a double-chain engineered therapeutic clostridial polypeptide. 2. The constructed therapeutic clostridium neurotoxin according to claim 1, where the modification contains a replacement with a lysine residue or an arginine residue. 3. An engineered therapeutic clostridium neurotoxin containing at least two amino acid modifications, where at least two of said amino acid modifications increase the isoelectric point (pI) of the engineered therapeutic neurotoxin to a value that is at least 0.2 units higher than the pI of another identical a clostridial neurotoxin not containing at least two of these amino acid modifications, wherein the at least two amino acid modifications are located in a clostridial toxin receptor binding domain (HC domain), wherein the engineered therapeutic clostridial neurotoxin is botulinum neurotoxin A (BoNT/A) having a pI of at least 6.6, wherein the at least two amino acid modifications include substitution of at least two amino acids selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083 , ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277, a lysine residue or an arginine residue, and wherein the engineered clostridial therapeutic neurotoxin is a double-chain engineered clostridial therapeutic neurotoxin where's the light chain and a heavy chain linked to each other by a disulfide bond, prepared by a method comprising providing a single chain engineered therapeutic clostridial neurotoxin comprising a polypeptide sequence having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NOs: 4, 6, 8 and 10 , or a single-chain engineered therapeutic clostridial neurotoxin encoded by a nucleotide sequence having at least 90% sequence identity with a nucleotide sequence selected from SEQ ID NOs: 3, 5, 7 and 9, and contacting the single-chain engineered therapeutic clostridial neurotoxin with a protease that cleaves the polypeptide at a cleavage site located between the light chain and the heavy chain, thereby converting the single-chain engineered therapeutic clostridial neurotoxin into a double-chain engineered therapeutic clostridial polypeptide. 4. The engineered therapeutic clostridial neurotoxin of any one of the preceding claims, wherein the double-strand engineered therapeutic clostridial neurotoxin has a safety factor of at least 8, wherein the safety factor is calculated as the dose of toxin required to produce a -10% change in body weight, measured as pg/mouse, and divided by ED50, measured -