HK1242703B - Cationic neurotoxins - Google Patents

Description

Cationic neurotoxins

The present invention relates to engineered clostridial toxins comprising at least one amino acid modification, and the use of such engineered clostridial toxins in medicine and therapy.

Bacteria in the genus clostridium (clostridium) produce highly potent and specific protein toxins that can poison neurons and other cells where they are delivered. Examples of such clostridial toxins include neurotoxins produced by clostridium tetani (c.tetani) (TeNT) and clostridium botulinum (c.botulin) (BoNT) serotypes a-G as well as those produced by clostridium pasteurianum (c.baratii) and clostridium butyricum (c.butyricum).

Some of the clostridial toxins are the strongest known toxins. For example, botulinum neurotoxin is a semi-Lethal Dose (LD) for mice ₅₀ ) Values range from 0.5 to 5ng/kg, depending on the serotype. Tetanus (tetanus)Both clostridial toxins and botulinum toxins act by inhibiting the function of the affected neurons, particularly the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic conduction in the peripheral nervous system, tetanus toxin acts in the central nervous system.

In nature, clostridial toxins are synthesized as single-chain polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains that are linked together by a disulfide bond. Cleavage occurs at specific cleavage sites (often referred to as activation sites) located between cysteine residues that provide interchain disulfide bonds. It is this double-stranded form that becomes the active form of the toxin. These two chains are referred to as the heavy chain (H-chain) (having a molecular weight of about 100 kDa) and the light chain (L-chain) (having a molecular weight of about 50 kDa). The H-chain comprises an N-terminal translocation element (H) _N Domain) and a C-terminal targeting element (Hc domain). The cleavage site is located between the L-chain and the translocation domain element. At H _C After binding of the domain to its target neuron and internalization of the bound toxin into the cell via endosome, H _N The domain translocates the L-chain across the endosomal membrane and into the cytoplasm, and the L-chain provides a protease function (also known as a non-cytotoxic protease). Non-cytotoxic proteases work by proteolytic cleavage of intracellular transporters (e.g., SNAP-25, VAMP, or syntaxin) known as SNARE proteins-see Gerald K (2002) "Cell and Molecular Biology" (4 th edition) John Wiley&Sons, Inc. The acronym SNARE derives from the term soluble NSF linked receptor, where NSF means N-ethylmaleimide-sensitive factor. SNARE proteins are required for vesicle fusion within cells and are therefore required for secretion of molecules from cells via vesicle transport. The protease function is a zinc-dependent endopeptidase activity and shows a high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cell, the non-cytotoxic protease is able to inhibit cellular secretion from the target cell. The L-chain protease of clostridial toxins is non-cytotoxic to cleave SNARE proteinsA protease.

In view of the ubiquitous nature of SNARE proteins, clostridial toxins such as botulinum toxin have been successfully used in a wide range of therapies.

For example, we refer to William J.Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc.,2004), which describes clostridial toxins, such as Clostridium Botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C ₁ The use of BoNT/D, BoNT/E, BoNT/F and BoNT/G, and clostridium tetani neurotoxin (TeNT) for inhibiting neuronal conduction in a number of therapeutic and cosmetic or aesthetic applications-for example, commercially available botulinum toxin products are currently approved as therapeutics for indications including focal spasms, upper limb spasms, lower limb spasms, cervical dystonia, blepharospasm, hemifacial spasm, axillary hyperhidrosis, chronic migraine, neurogenic detrusor overactivity, glabellar line, and severe lateral canthal line. In addition, clostridial toxin therapies are described for the treatment of neuromuscular disorders (see US 6,872,397); for the treatment of uterine disorders (see US 2004/0175399); for the treatment of ulcers and gastroesophageal reflux disease (see US 2004/0086531); for the treatment of dystonia (see US 6,319,505); for the treatment of ocular disorders (see US 2004/0234532); for the treatment of blepharospasm (see US 2004/0151740); for the treatment of strabismus (see US 2004/0126396); for the treatment of pain (see US 6,869,610, US 6,641,820, US 6,464,986 and US 6,113,915); for the treatment of fibromyalgia (see US 6,623,742, US 2004/0062776); for the treatment of lower back pain (see US 2004/0037852); for the treatment of muscle damage (see US 6,423,319); for the treatment of sinus headache (see US 6,838,434); for the treatment of tension headaches (see US 6,776,992); for the treatment of headache (see US 6,458,365); for reducing migraine headaches (see US 5,714,469); for the treatment of cardiovascular diseases (see US 6,767,544); for the treatment of neurological disorders such as parkinson's disease (see US 6,620,415, US 6,306,403); for the treatment of neuropsychiatric disorders (see US 2004/0180061, US 2003/0211121); for the treatment of endocrine disorders (see US 6,827,931); for the treatment of thyroid disorders (see US 6,740,321); used for treating cholinergic shadowA loud sweat gland disorder (see US 6,683,049); for the treatment of diabetes (see US 6,337,075, US 6,416,765); for the treatment of pancreatic disorders (see US 6,261,572, US 6,143,306); for the treatment of cancer such as bone tumors (see US 6,565,870, US 6,368,605, US 6,139,845, US 2005/0031648); for the treatment of ear disorders (see US 6,358,926, US 6,265,379); for the treatment of autonomic disorders such as gastrointestinal muscle disorders and other smooth muscle dysfunction (see US 5,437,291); for the treatment of skin lesions associated with skin cell proliferation disorders (see US 5,670,484); for the control of neurogenic inflammatory disorders (see US 6,063,768); for reducing hair loss and stimulating hair growth (see US 6,299,893); for treatment of the angle of the mouth facing downwards (see US 6,358,917); for reducing appetite (see US 2004/40253274); for treatment of teeth and dental surgery (see US 2004/0115139); for the treatment of neuromuscular disorders and conditions (see US 2002/0010138); for the treatment of various disorders and conditions and associated pain (see US 2004/0013692); for the treatment of disorders caused by mucus hypersecretion such as asthma and COPD (see WO 00/10598); and for the treatment of non-neuronal disorders such as inflammation, endocrine disorders, exocrine disorders, immune disorders, cardiovascular disorders, bone disorders (see WO 01/21213). All of the above publications are incorporated herein by reference in their entirety.

The use of non-cytotoxic proteases such as clostridial toxins (e.g., BoNT and TeNT) in the therapeutic and cosmetic treatment of humans and other mammals is expected to expand into an increasingly broad range of diseases and conditions that can benefit from the properties of these toxins.

To avoid systemic neurologic effects, many clostridial toxin therapies utilize direct administration of a clostridial toxin therapeutic agent to a given target site (e.g., a target tissue). One problem when administering clostridial toxin-based therapeutics in this manner is the spread of the toxin away from the site of administration into the surrounding tissue or systemic circulation. The diffusion of toxins away from target tissues is thought to be responsible for unwanted side effects, which in extreme cases may be life threatening. This may be a particular concern when clostridial toxin therapeutics (e.g., BoNT therapeutics) are used at high doses, concentrations, and injection volumes. For commercial BoNT/a therapeutics, side effects that have been reported to be associated with this problem include: weakness, general muscle weakness, double vision, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence and dyspnea. Swallowing and breathing difficulties can be life-threatening and death associated with the spread of toxin action has been reported.

Thus, there is a need in the art for clostridial toxins that have increased tissue retention properties at the site of administration and thus exhibit reduced diffusion away from the site of administration compared to known clostridial toxins.

The present invention solves the above problems by providing engineered clostridial toxins, as specified in the claims.

In one aspect, the invention provides an engineered clostridial toxin comprising at least one (e.g., at least one, two or three) 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 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of an otherwise identical clostridial toxin lacking the at least one amino acid modification, and wherein the at least one amino acid modification is not located in the clostridial toxin binding domain (H), pI _C Domains).

In one embodiment, "not located in a clostridial toxin binding domain (H) _C ) By "in the domain of the structure" is meant that the at least one amino acid modification is located in the H of a clostridial toxin _N Domains or the light chain of clostridial toxins.

In one embodiment, the invention provides an engineered clostridial toxin comprising at least one (e.g., at least one, two or three) amino acid modification, wherein said 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 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0) higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification.9 or 1) pI units, and wherein the at least one amino acid modification is not located in the translocation domain (H) of a clostridial toxin _N Domains).

In another embodiment, the invention provides an engineered clostridial toxin comprising at least one (e.g., at least one, two or three) amino acid modification, wherein said 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 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification, and wherein said at least one amino acid modification is located in the clostridial toxin light chain.

In one embodiment, wherein the at least one amino acid modification is in the clostridial toxin light chain, the at least one amino acid modification does not introduce an E3 ligase recognition motif into the clostridial toxin light chain. Thus, in one embodiment, the light chain of the engineered clostridial toxins of the present invention does not comprise an E3 ligase recognition motif.

As used above, the term "E3 ligase recognition motif refers to a modification of the light chain that results in accelerated degradation of the neurotoxin polypeptide by endogenous proteasome degradation pathways present in the individual to whom the neurotoxin is applied. The "E3 ligase recognition" motif is a structural motif that allows recognition of the motif by E3 ligase (also known as E3 ubiquitin ligase; thus, the "E3 ligase recognition motif" may also be known as the "E3 ubiquitin ligase recognition motif") and binding of the motif. The recognition motif for the E3 ligase is familiar to those skilled in the art.

Examples of E3 ligase recognition motifs include the following sequences (where "X" may represent any naturally occurring amino acid):

other examples of E3 ligase recognition motifs include: ETFSDLWKLLPE (SEQ ID NO: 20), TSFAEYWNLLSP (SEQ ID NO: 21), LTFEHYWAQLTS (SEQ ID NO: 22), LTFEHWWAQLTS (SEQ ID NO: 23), LTFEHSWAQLTS (SEQ ID NO: 24), ETFEHNWAQLTS (SEQ ID NO: 25), LTFEHNWAQLTS (SEQ ID NO: 26), LTFEHWWASLTS (SEQ ID NO: 27), LTFEHWWSSLTS (SEQ ID NO: 28), LTFTHWWAQLTS (SEQ ID NO: 29), ETFEHWWAQLTS (SEQ ID NO: 30), LTFEHWWSQLTS (SEQ ID NO: 31), LTFEHWWAQLLS (SEQ ID NO: 32), ETFEHWWSQLLS (SEQ ID NO: 33), RFMDYWEGL (SEQ ID NO: 34), MPRFMDYWEGLN (SEQ ID NO: 35), SQETFSDLWKLLPEN (SEQ ID NO: 36), and LTFEHNWAQLEN (SEQ ID NO: 37).

In one embodiment, wherein the at least one amino acid modification is located in the light chain of the clostridial toxin, the at least one amino acid modification does not introduce a MDM 2E 3 ligase recognition motif into the light chain of the clostridial toxin. Thus, in one embodiment, the light chain of the engineered clostridial toxins of the present invention does not comprise a MDM 2E 3 ligase recognition motif.

In one embodiment, wherein the at least one amino acid modification is in the light chain of the clostridial toxin, the engineered clostridial toxin comprising no amino acid modification at the N-terminal proline.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, and wherein the at least one amino acid modification is located in the clostridial toxin light chain, the engineered BoNT/E does not comprise a substitution with a lysine at any of the following amino acid positions: q53, N72, N378, N379, R394, T400.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, and wherein said at least one amino acid modification is located in the clostridial toxin light chain, said engineered BoNT/E does not comprise a substitution with lysine at any one of the following amino acid positions: q53, N72, N378, N379, T400.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, and wherein said at least one amino acid modification is located in the clostridial toxin light chain, said engineered BoNT/E does not comprise a substitution with a lysine at any three of the following amino acid positions: q53, N72, N378, N379, R394, T400.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, and wherein said at least one amino acid modification is located in the clostridial toxin light chain, said engineered BoNT/E does not comprise a substitution with a lysine at any three of the following amino acid positions: q53, N72, N378, N379, T400.

In one embodiment, optionally wherein the at least one amino acid modification is located in the light chain of the clostridial toxin, the engineered clostridial toxin is not BoNT/E.

Engineered clostridial toxins of the invention do not comprise toxin H _C Any amino acid modification in a domain. Thus, in the engineered clostridial toxins of the present invention, the at least one amino acid modification is not located in the clostridial toxin H _C A domain of structure.

In one embodiment, wherein the at least one amino acid modification is in the light chain of the clostridial toxin, the at least one amino acid modification does not include the substitution of an amino acid residue with a lysine residue.

In one embodiment, wherein the engineered clostridial toxin is an engineered clostridial toxin as described above, the at least one amino acid modification comprises substitution of an acidic amino acid residue or an uncharged amino acid residue with a lysine or arginine residue.

In one embodiment, wherein the engineered clostridial toxin is an engineered clostridial toxin as described above, the at least one amino acid modification comprises substitution of an acidic amino acid residue or an uncharged amino acid residue with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is an engineered clostridial toxin as described above, wherein said at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 0.4 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 0.5 pI units higher than the pI of an otherwise identical clostridial toxin lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 0.6 pI units higher than the pI of an otherwise identical clostridial toxin lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 0.8 pI units higher than the pI of an otherwise identical clostridial toxin lacking the at least one amino acid modification. In one embodiment, the at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 1 pI unit higher than the pI of an otherwise identical clostridial toxin lacking the at least one amino acid modification.

In certain embodiments, the engineered clostridial toxin comprises at least 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 amino acid modifications.

In certain embodiments, the at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 2,3, 4 or 5 pI units higher than the pI of an otherwise identical clostridial toxin lacking the at least one amino acid modification.

In certain embodiments, the engineered clostridial toxin comprises at least 3 amino acid modifications, and said at least 3 amino acid modifications increase the pI of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least 3 amino acid modifications.

In certain embodiments, the engineered clostridial toxin comprises at least 5 amino acid modifications, and said at least 5 amino acid modifications increase the pI of the engineered clostridial toxin to a value that is at least 0.5 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least 5 amino acid modifications.

The present inventors have found that by increasing the pI of a clostridial toxin, for example by at least 0.2 pI units or 0.5 pI units or 1 pI unit (by introducing at least one amino acid modification into the clostridial toxin protein), the resulting engineered clostridial toxin advantageously exhibits increased tissue retention properties and reduced diffusion away from the site of administration, while retaining the ability of target cell binding, translocation and cleavage of the target SNARE protein. Thus, transmission of the clostridial toxin from the site of administration is significantly reduced as compared to an otherwise identical clostridial toxin lacking the at least one amino acid modification.

The engineered clostridial toxins of the invention are suitable for use in any of the above-described treatments, and may advantageously exhibit reduced or no side effects as compared to the use of known clostridial toxin therapeutics.

The increased tissue retention properties of the engineered clostridial toxins of the present invention also provide increased efficacy and/or duration of action, and can allow for the use of reduced doses (or increased doses without any additional side effects) compared to known clostridial toxin therapeutics, providing further advantages.

Thus, in one embodiment, the engineered clostridial toxins of the invention have increased potency, increased tissue retention, and/or increased duration of action compared to the corresponding unmodified clostridial toxins.

As discussed in more detail below, the increase in pI provided by the at least one amino acid modification means that the engineered clostridial toxin of the invention has a net charge at a given pH that is more positive than the net charge on an otherwise identical clostridial toxin lacking the at least one amino acid modification. Without wishing to be bound by any one theory, the inventors believe that this increased positive charge allows the engineered clostridial toxins of the invention to exhibit longer tissue retention times at the site of administration due to the favorable electrostatic interaction between the engineered clostridial toxin and anionic extracellular components (e.g., cell membrane and heparin sulfate proteoglycan) at the site of administration. These improved electrostatic interactions serve to reduce the diffusion of the engineered clostridial toxin away from the site of administration, thereby improving tissue retention.

For example, the improved tissue retention properties of the engineered clostridial toxins of the present invention can allow (i) higher doses to enter individual muscles, such as the sternocleidomastoid muscle, without spreading into nearby neck muscles causing dysphagia, and (ii) higher total doses (for all muscles) in a single treatment, without spreading into the circulation, causing systemic effects such as dyspnea. Benefits for the patient may include more effective treatment of large muscles (e.g., sternocleidomastoid), increased opportunity to inject several different muscles during each treatment, and potentially longer effective treatment duration due to higher dosing (longer duration is necessary before re-treatment).

In one embodiment, the engineered clostridial toxins of the invention have a positive net charge in use (e.g., when the engineered clostridial toxin is located at a desired site of administration in a tissue in use).

The isoelectric point (pI) is a characteristic of a given protein. As is well known in the art, a protein is composed of a sequence of specific amino acids (also referred to as amino acid residues when in a protein). Each amino acid in the standard set of 20 amino acids has a different side chain (or R group), meaning that in one protein, each amino acid residue exhibits different chemical properties such as charge and hydrophobicity. These properties may be influenced by the surrounding chemical environment (e.g., temperature and pH). The overall chemical characteristics of a protein will depend on the sum of these multiple factors.

Certain amino acid residues (described in detail below) have side chains capable of ionization, which may exhibit a charge, depending on the surrounding pH. At a given pH, whether such a side chain is charged or not depends on the pKa of the relevant moiety capable of ionization, where pKa is the negative logarithm of the dissociation constant (Ka) for the separation of a given proton of the acid from a conjugate base.

For example, acidic residues such as aspartic acid and glutamic acid have pendant carboxylic acid groups with pKa values of about 4.1 (the exact pKa value may depend on temperature, ionic strength and the microenvironment of the group capable of ionization). Thus, these side chains exhibit a negative charge at pH7.4 (often referred to as "physiological pH"). At low pH, these side chains will 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 about 10-12. These side chains thus show a positive charge at ph 7.4. At high pH, these side chains will become deprotonated and lose their charge.

Thus, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and the pH of the environment. Changing the pH of the environment changes the overall charge on the protein. Thus, for each protein, there is a given pH at which the number of positive and negative charges are equal and the protein exhibits no net charge overall. This point is called the isoelectric point (pI). Isoelectric point is a standard concept in protein biochemistry and will be familiar to the skilled person.

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

Methods for determining the pI of a protein are known in the art and will be familiar to the skilled person. For example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pI"). Such calculations may be performed using computer programs known in the art; preferred exemplary computer programs for calculating the pI value include Protein Call Calculator from Scripps Research Institute and computer pI/MW tool from ExPASy. The same calculation technique/program should be used to compare pI values between different molecules.

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

In the present specification, unless otherwise specified, "pI" means "calculated pI".

By varying the number of basic groups and/or acidic groups displayed on the surface of the protein, the pI of the protein can be increased or decreased. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI can be provided by decreasing the number of acidic residues or by increasing the number of basic residues. Such amino acid modifications are discussed in more detail below.

Native (unmodified) clostridial toxins have a pI of about 5-6. Thus, at pH7.4, the native botulinum toxin has a negative net charge. For example, the pI of BoNT/A is 6.4, and the BoNT/A molecule has a net charge of-8 at pH 7.4. These pI values were calculated as described above.

TABLE 1

Clostridial toxins	pI
		BoNT/A	6.4
BoNT/B	5.3
		BoNT/C 1	5.5
BoNT/D	5.5
		BoNT/E	6.0
BoNT/F	5.6
		BoNT/G	5.2
TeNT	5.8

As noted above, in one embodiment, the engineered clostridial toxins of the invention comprise at least one amino acid modification, wherein said 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 an otherwise identical clostridial toxin lacking said at least one amino acid modification.

Thus, in the context of the present invention, an increase of 0.2 units in pI in the context of an engineered BoNT/a clostridial toxin would be an increase in pI from 6.4 to 6.6.

As noted above, in one embodiment, the engineered clostridial toxins of the invention comprise at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 1 pI unit higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification.

Thus, in the context of the present invention, an increase of 1 unit in pI in the context of an engineered BoNT/a clostridial toxin would be an increase in pI from 6.4 to 7.4.

In one embodiment, the engineered clostridial toxin has a pI of at least 5.5.

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

In one embodiment, the engineered clostridial toxin has a pI of at least 6.5.

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

In one embodiment, the engineered clostridial toxin has a pI of 6.5 to 9.5 (e.g., a pI of 6.5 to 7.5).

As described above, the engineered clostridial toxins of the present invention have increased tissue retention properties, which also provide increased efficacy and/or duration of action, and can allow for reduced dosages to be used (or allow for increased dosages without any additional effects) compared to known clostridial toxin therapeutics. One way in which these advantageous properties, which represent an increase in therapeutic index, can be defined is in terms of the safety ratio of the engineered clostridial toxin. In this regard, by measuring the percentage of weight loss in the relevant animal model (e.g., mice, where weight loss is detected within 7 days of administration), the undesirable effects of clostridial toxins (caused by diffusion of the toxin away from the site of administration) can be experimentally assessed. Conversely, the desired on-target effect of clostridial toxins can be assessed experimentally by a toe abduction score (DAS) analysis (muscle paralysis measure). DAS analysis can be performed by injecting 20. mu.L of clostridial toxin formulated in gelatin phosphate buffer into the mouse gastrocnemius/soleus complex, and then assessing the toe abduction score by the method using Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS analysis, the mouse is suspended briefly by the tail to elicit a characteristic panic response, in which the mouse stretches its hind limbs and abducts its hind toes. After injection of clostridial toxin, different degrees of toe abduction were scored according to the five-point scale (0 ═ normal to 4 ═ toe abduction and greatest reduction in leg extension).

The safety ratio of clostridial toxins can then be expressed as the ratio between the amount of toxin required to reduce body weight by 10% (measured at peak effect within the first 7 days after administration in mice) and the amount of toxin required to score a DAS score of 2. Thus, a high safety ratio score is desirable and indicates a toxin that is effective in paralyzing the target muscle and has little undesirable off-target effect. The engineered toxins of the present invention have a safety ratio that is higher than that of comparable unmodified (native) botulinum toxins.

Thus, in one embodiment, an engineered clostridial toxin of the invention has a safety ratio of at least 8 (e.g., at least 8,9, 10, 15, 20, 25, 30, 35, 40, 45 or 50), wherein the safety ratio is calculated by: dose of toxin required to change body weight-10% (pg/mouse) divided by DASED ₅₀ (pg/mouse) [ ED ₅₀ Dose required to produce a DAS score of 2]。

In one embodiment, the engineered clostridial toxins of the present invention have a safety ratio of at least 10. In one embodiment, the engineered clostridial toxins of the present invention have a safety ratio of at least 15.

The engineered clostridial toxins of the invention comprise at least one amino acid modification. The at least one amino acid modification increases the pI of the clostridial toxin, as described 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 modifications may be made by substituting one amino acid in the sequence for another (i.e., substitution), by inserting a new amino acid into the sequence, or by deleting amino acids from the sequence. Amino acids inserted into the amino acid sequence of a protein are also referred to as amino acid residues.

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

table 2:

the following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive) and lysine (positive).

At ph7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.

The following amino acids are considered to be uncharged polar (meaning that they are capable of participating in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan.

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

An increase in the pI of a clostridial toxin can be achieved by introducing into the clostridial toxin one or more amino acid modifications that increase the ratio of positive to negative charges in the clostridial toxin.

In one embodiment, the at least one amino acid modification is selected from: amino acid substitutions, amino acid insertions, and amino acid deletions.

In amino acid substitutions, the amino acid residues that form part of the clostridial toxin amino acid sequence are replaced with different amino acid residues. The replacement amino acid residue may be one of the 20 standard amino acids described above.

Alternatively, in an amino acid substitution, the substituted amino acid may be a non-standard amino acid (an amino acid that is not part of the standard set of 20 amino acids described above). For example, the replacement amino acid can be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine). Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an e.coli (e.coli) auxotrophic expression host.

In amino acid insertions, additional amino acid residues (one amino acid not normally present) are incorporated into the clostridial toxin amino acid sequence, thereby increasing the total number of amino acid residues in the sequence. In the amino acid deletion, amino acid residues are removed from the clostridial toxin amino acid sequence, thereby reducing the total number of amino acid residues in the sequence.

Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. For example, amino acid modifications can be introduced by modification of the DNA sequence encoding the clostridial toxin. This can be achieved using standard molecular cloning techniques, for example by site-directed mutagenesis using a polymerase, replacing the original coding sequence with a short strand of DNA (oligonucleotide) encoding the desired amino acid, or by inserting/deleting parts of the gene using a variety of enzymes, such as ligases and restriction endonucleases. Alternatively, the modified gene sequence can be chemically synthesized.

In one embodiment, the at least one amino acid modification is selected from: the acidic amino acid residue is substituted with a basic amino acid residue; the acidic amino acid residue is substituted with an uncharged amino acid residue; the uncharged amino acid residue is substituted with a basic amino acid residue; insertion of a basic amino acid residue; and deletion of acidic amino acid residues.

In a preferred embodiment, the at least one amino acid modification is a substitution, which advantageously keeps the number of amino acid residues in the clostridial toxin the same. In one embodiment, the substitution is selected from: the acidic amino acid residue is substituted with a basic amino acid residue, the acidic amino acid residue is substituted with an uncharged amino acid residue and the uncharged amino acid residue is substituted with a basic amino acid residue. In one embodiment, the basic amino acid residue is a lysine residue or an arginine residue. In one embodiment, the basic amino acid residue is a lysine residue. In one embodiment, the basic amino acid residue is an arginine residue. In one embodiment, wherein the substitution is of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced with asparagine and glutamic acid is replaced with glutamine).

In another preferred embodiment, the at least one amino acid modification is the substitution of an acidic amino acid residue by a basic amino acid residue or the substitution of an uncharged amino acid residue by a basic amino acid residue. In one embodiment, the basic amino acid residue is a lysine residue. In one embodiment, the basic amino acid residue is an arginine residue.

Engineered clostridial toxins of the invention can comprise more than one amino acid modification. Thus, in one embodiment, the engineered clostridial toxin (as described above) comprises 1 to 90 amino acid modifications (e.g., 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 3 to 50, 3 to 40,3 to 30, 4 to 40, 4 to 30, 5 to 40, 5 to 30, or 10 to 25 amino acid modifications). In one embodiment, the engineered clostridial toxin (described above) comprises 1 to 20 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises 1 to 10 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises from 2 to 20 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises 2 to 15 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises 2 to 10 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises from 3 to 20 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises 3 to 15 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises 3 to 10 amino acid modifications. In one embodiment, the engineered clostridial toxin (described above) comprises from 4 to 40 amino acid modifications. In one embodiment, the engineered clostridial toxin comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid modifications. In one embodiment, the engineered clostridial toxin comprises at least 3 amino acid modifications (e.g., at least 3 amino acid substitutions). In one embodiment, the engineered clostridial toxin comprises at least 4 amino acid modifications (e.g., at least 4 amino acid substitutions). Each of the amino acid modifications is an amino acid modification as described above. Thus, each of the amino acid modifications contributes to an increase in the pI of the engineered clostridial toxin (as compared to the pI of an otherwise identical clostridial toxin lacking the amino acid modification).

Any is not located in the clostridial toxin binding domain (H) _C Domain) can be modified as described above, provided that the modification results in an increase in the pI of the clostridial toxin (as described above). However, the present inventors have identified a subset of clostridial toxin amino acids that are particularly suitable for use as a target for modification.

Preferred target amino acids may have certain properties. For example, preferred target amino acids may: (i) is a surface exposed amino acid; (ii) located outside of the clostridial toxin protein secondary structure; (iii) located in a region of the clostridial toxin protein that is not essential for protein function; (iv) is an amino acid whose identity is not retained between the types, subtypes or serotypes of clostridial toxins; (iv) is an amino acid whose modification does not result in the desired ubiquitination site; or (v) is any combination of the foregoing.

As noted above, the engineered clostridial toxins of the present invention are characterized by being located in clostridial toxin H _N A translocation domain or one or more amino acid modifications located in the clostridial toxin light chain.

Examples of clostridial toxin light chain reference sequences include:

clostridium botulinum neurotoxin type a: amino acid residues 1 to 448

Botulinum neurotoxin type B: amino acid residues 1-440

Clostridium botulinum C ₁ Type i neurotoxin: 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 to 439

Botulinum neurotoxin type G: amino acid residues 1-441

Clostridium tetani neurotoxin: amino acid residues 1-457.

Clostridial toxin H _N Examples of domain reference sequences include:

clostridium botulinum neurotoxin type a: amino acid residue 449-

Botulinum neurotoxin type B: amino acid residue 443-

Clostridium botulinum C ₁ Type i neurotoxin: amino acid residue 450-866

Clostridium botulinum neurotoxin type D: amino acid residue 449-

Botulinum neurotoxin type E: amino acid residue 455-845

Botulinum neurotoxin type F: amino acid residue 450-864

Botulinum neurotoxin type G: amino acid residue 449-

Clostridium tetani neurotoxin: amino acid residue 456-879.

Clostridial toxin H _C Examples of domain reference sequences include:

clostridium botulinum neurotoxin type a: amino acid residue 872-

Botulinum neurotoxin type B: amino acid residue 863-1291

Clostridium botulinum C ₁ Type i neurotoxin: amino acid residue 867-1291

Clostridium botulinum neurotoxin type D: amino acid residue 872-1276

Botulinum neurotoxin type E: amino acid residue 846-1252

Botulinum neurotoxin type F: amino acid residue 865-1278

Botulinum neurotoxin type G: amino acid residue 872-1297

Clostridium tetani neurotoxin: amino acid residues 880-1315.

The above reference sequences should be considered as guidelines, as slight variations may occur depending on the sub-serotype. For example, US 2007/0166332 (the entire contents of which are incorporated herein by reference) lists slightly different clostridial sequences:

light chain:

clostridium botulinum neurotoxin type a: amino acid residues M1-K448

Botulinum neurotoxin type B: amino acid residues M1-K441

Clostridium botulinum C ₁ Type i neurotoxin: 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

Clostridium botulinum type G neurotoxin: amino acid residues M1-K446

Clostridium tetani neurotoxin: amino acid residues M1-A457.

H _N Domain (b):

clostridium botulinum neurotoxin type a: amino acid residues A449-K871

Botulinum neurotoxin type B: amino acid residues A442-S858

Clostridium botulinum C ₁ Type i neurotoxin: amino acid residues T450-N866

Clostridium botulinum neurotoxin type D: amino acid residues D446-N862

Botulinum neurotoxin type E: amino acid residues K423-K845

Botulinum neurotoxin type F: amino acid residues A440-K864

Clostridium botulinum type G neurotoxin: amino acid residues S447-S863

Clostridium tetani neurotoxin: amino acid residues S458-V879.

H _C Domain (b):

clostridium botulinum neurotoxin type a: amino acid residues N872-L1296

Botulinum neurotoxin type B: amino acid residues E859-E1291

Clostridium botulinum C ₁ Type i neurotoxin: amino acid residue N867-E1291

Clostridium botulinum neurotoxin type D: amino acid residues S863-E1276

Botulinum neurotoxin type E: amino acid residues R846-K1252

Botulinum neurotoxin type F: amino acid residues K865-E1274

Botulinum neurotoxin type G: amino acid residues N864-E1297

Clostridium tetani neurotoxin: amino acid residues I880-D1315.

In one embodiment, wherein the at least one amino acid modification is located in the translocation domain (H) of the clostridial toxin _N Domain), the at least one amino acid modification is not located in a clostridial toxin belt region (belt region). Clostridial toxin band regions (determined by visual inspection of structure and model) are defined as follows:

clostridium botulinum neurotoxin type a: amino acid residue 492-545

Botulinum neurotoxin type B: amino acid residue 472-

Clostridium botulinum C ₁ Type i neurotoxin: amino acid residue 494-

Botulinum neurotoxin type D: amino acid residue 489-539

Botulinum neurotoxin type E: amino acid residue 466-

Botulinum neurotoxin type F: amino acid residue 485-

Clostridium botulinum type G neurotoxin: amino acid residue 489-538

Clostridium tetani neurotoxin: amino acid residue 506 and 556.

In one embodiment, the at least one amino acid modification (as described above) is a modification of a surface exposed amino acid residue. Surface exposed amino acid residues are those that are present on the exterior of the folded protein and are therefore accessible to the surrounding solvent, as opposed to those that are located in the interior of the folded protein. The degree of surface exposure of an amino acid residue, and therefore its exposure to surrounding solvent, depends on its position in the folded protein and also on the conformation adopted by the protein. Thus, modification of amino acid residues with a high degree of surface exposure may have a greater effect on the isoelectric point of the protein than modification of amino acid residues with a low degree of surface exposure. Methods for determining the degree of surface exposure of amino acid residues are known in the art. For example, the computer program ArealMol (part of the CCP4 computer program suite) can be used to calculate the degree of surface exposure of amino acid residues in a given protein. Surface exposed amino acid residues can also be identified by visual inspection of the protein crystal structure (e.g., as provided by X-ray crystallography). In one embodiment, the surface exposed amino acid residues have a total AreaMol value of at least 40.

In one embodiment, the at least one amino acid modification comprises a modification of an amino acid residue selected from the group consisting of: aspartic acid residues, glutamic acid residues, histidine residues, asparagine residues, glutamine residues, serine residues, threonine residues, alanine residues, glycine residues, valine residues, leucine residues, and isoleucine residues. The inventors have identified that the amino acid residues in this group represent particularly suitable targets for modification according to the invention.

In one embodiment, wherein the amino acid modification comprises a modification of an amino acid residue selected from the group consisting of: an aspartic acid residue, a glutamic acid residue, a histidine residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, a valine residue, a leucine residue, and an isoleucine residue (as described above), the amino acid residue being substituted with a lysine residue or an arginine residue. In one embodiment, the amino acid residue is substituted with an arginine residue. Thus, in one embodiment, a negatively charged residue, a polar residue, or an uncharged residue is substituted with a positively charged residue, thereby increasing the positive to negative ratio and increasing the pI of the clostridial toxin.

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

In one embodiment, the engineered clostridial toxin is BoNT/A. The reference BoNT/A sequence has UniProtKB accession number P10845. In one embodiment, wherein the engineered clostridial toxin is BoNT/A and the engineered clostridial toxin is BoNT/A1.

The present inventors have identified a toxin H representative of the Clostridium BoNT/A toxin _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/a, said engineered BoNT/a comprises at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or all 55) amino acid modification selected from the group consisting of: d474, N476, D484, N486, I487, E488, a489, a490, E491, D546, E558, E560, H561, I566, L568, N570, S571, L577, N578, a597, E599, a601, E620, V621, T623, D625, T631, N645, L647, D650, D651, I668, E670, a672, V675, S683, I685, a686, N687, N752, Q753, T755, E757, E758, N760, N761, I762, N763, D825, I831, G832, T847, D848, and D858; and the one or more amino acid modifications 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 of an otherwise identical BoNT/a lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/a, said engineered BoNT/a comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, or all 17) amino acid selected from the group consisting of: n476, S564, N578, E599, L647, D650, D651, V675, I685, N687, T755, E757, N761, N763, I831, T847 and I849; and the one or more amino acid modifications 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 of an otherwise identical BoNT/a lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/a, said engineered BoNT/a comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, or all 6) amino acid selected from the group consisting of: s564, L647, D650, D651, T847, and I849; and the one or more amino acid modifications 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 of an otherwise identical BoNT/a lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/a, said engineered BoNT/a comprises a modification of at least one (e.g., at least 1, 2,3, 4, 5, or all 6) amino acid selected from the group consisting of: n476, N763, N687, E599, I831, and N761; and the one or more amino acid modifications 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 of an otherwise identical BoNT/a lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/a, said engineered BoNT/a comprising a modification of at least one (e.g., at least 1, 2,3, 4, or all 5) amino acid selected from the group consisting of: n578, V675, I685, T755 and E757; and the one or more amino acid modifications 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 of an otherwise identical BoNT/a lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

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

The present inventors have identified a toxin H representative of Clostridium BoNT/B _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/B comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, or all 40) amino acid selected from the group consisting of: v443, G444, D453, S468, D533, E534, N535, T545, L548, D549, I550, D552, S557, L564, S566, N582, V584, N609, L619, N632, E633, G637, a646, I655, E657, V662, E669, S670, I672, D673, N739, I740, N748, N750, I818, G819, T834, I842, N845 and S858; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/B to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/B lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

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

The present inventors have identified that a representation of BoNT/C ₁ Clostridial toxin H _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/C ₁ Said engineered BoNT/C ₁ A modification comprising at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or all 50) amino acid selected from: l451, D452, C453, E455, V472, T474, D475, L478, N483, E484, E485, E487, I489, L555, S556, D557, N558, E560, D561, E569, N574, S575, T584, G592, Q594, G596, D617, N640, S641, V642, G645, N646, E661, E665, T667, a670, S678, V680, Q681, E682, S750, G751, S759, Q760, V826, G827, N842, T843, N847 and N853; and said one or more amino acid modifications engineer a BoNT/C ₁ To a value that is greater than an otherwise identical BoNT/C lacking said one or more amino acid modifications ₁ 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 pI of (a) pI of (b). In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

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

The present inventors have identified a representative BoNT/D clostridial toxin H _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/D, said engineered BoNT/D comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or all 62) amino acid selected from the group consisting of: q469, E470, E473, N474, D479, E480, N482, V483, Q484, N485, S487, D488, S552, N553, N554, V555, E556, N557, I558, L560, T562, S563, V564, G569, S571, N572, G588, Q590, T614, D616, S619, S622, N636, S637, L639, G641, N642, E657, E558, T663, a666, V669, S674, I676, Q677, E678, S746, G747, D749, E751, N752, I753, Q756, N818, V822, G823, E837, N838, T839, N843, N849, and N850; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/D to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/B lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is BoNT/E. The reference BoNT/E sequence has UniProtKB accession number Q00496. In one embodiment, wherein the engineered clostridial toxin is BoNT/E and the engineered clostridial toxin is BoNT/E3.

The present inventors have identified a representative clostridial toxin H that is BoNT/E _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, said engineered BoNT/E comprises at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or all 52) modification of an amino acid selected from the group consisting of: d474, N476, E479, E480, D484, N486, I487, E488, a489, a490, E491, E492, L496, D497, Q500, Q501, L504, N507, D509, N510, N514, S516, E518, Q527, L530, N533, I534, E535, N539, Y548, I566, L568, D589, a597, E599, a601, L604, Y612, E620, N645, L647, Y648, D651, E737, E741, Y803, Y824, D825, G831, I831, G832 and D835; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/E lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

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

The present inventors have identified a representative BoNT/F clostridial toxin H _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/F, said engineered BoNT/F comprises at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or all 86) modification of an amino acid selected from the group consisting of: n463, E464, N468, T469, D474, D475, T476, T477, N478, N482, N485, N495, I499, Q501, I502, Q505, T506, N508, T509, V511, D513, D521, S522, S526, E527, I528, E529, V534, D535, L536, E549, G550, T552, N553, S558, E566, E567, S568, V586, H587, Q608, D613, a616, D617, S619, N630, N633, N639, E654, V656, E658, L660, T663, L665, V666, S671, I673, G674, S675, S676, E677, N678, T746, N751, L753, E754, E756, N759, I75821, N759, I673, S761, S851, I761, S851, I849, S851, L849, L851, L761, S851, L849, L851, L751, L845, L851, L849, L78, L851, L78, S851, L78, S851, L78, S851, L78, S851, L78, S851, L78, S851, L78, L851, S851, L78, S851, L78, S82, S851, L78, S851, L78, S82, L78, S82, S851, S82, S851, S82, S18, L78, S82, S18, S82, S851, S82, S18, S851, S18, S82, S18, L78, S18, S851, S18, S851, L78, S851, L78, S82, S851, L78, S851, S82, L78, S851, S82, L78, S851, S82, L78, S82, S851, L78, and S851, L78, L; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/F to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/F lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

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

The present inventors have identified a toxin H that is representative of Clostridium BoNT/G _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/G, said engineered BoNT/G comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, or all 36) amino acid selected from the group consisting of: n480, Q482, N483, N484, T485, E487, D540, N562, N570, N571, N572, T588, V589, T615, D621, N637, E638, E642, N643, I660, E662, I667, E674, S675, V677, G678, N679, S747, N755, D757, L823, D839, I841, D844, S846, and L847; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/G to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/G lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

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

The present inventors have identified a representative toxin H of Clostridium tett _N Certain amino acids that are preferred targets for amino acid modifications in a domain.

In one embodiment, wherein the engineered clostridial toxin is a TeNT comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or all 49) amino acid selected from the group consisting of: a457, S458, L459, D461, L462, E486, E487, Q490, D491, N497, N504, D557, T571, T572, L573, Q574, N580, S581, N588, S589, T590, S598, Q605, G606, Q608, T631, I633, S640, Q655, E658, G659, N660, E675, I677, E679, T681, V684, a691, E692, S694, T695, Q696, a772, D773, E774, S862, N866, L867 and D868; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered TeNT to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical TeNT lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modifications in the clostridial toxin light chain of BoNT/a.

In one embodiment, wherein the engineered clostridial toxin is BoNT/a, said engineered BoNT/a comprises a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, or all 28) amino acid selected from the group consisting of: n5, Q7, N9, D12, N15, Q31, D58, N60, D74, N82, T122, D124, E126, Q139, D141, E281, L284, S295, Q311, D326, D334, N377, TYR387, N394, N396, N410, M411, and N418; and the one or more amino acid modifications 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 of an otherwise identical BoNT/a lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modifications in the clostridial toxin light chain of BoNT/B.

In one embodiment, wherein the engineered clostridial toxin is BoNT/B, said engineered BoNT/B comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or all 41) amino acid selected from the group consisting of: n6, N7, N9, N11, D12, N16, N17, N18, D41, E48, E57, N60, D75, D77, N80, E127, N130, N144, E147, E149, E185, N216, D245, E253, N316, D333, E335, D341, N385, D388, N389, E390, E395, E396, D402, D404, E406, E408, Q419, E423 and E427; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/B to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/B lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present inventors have identified representatives of BoNT/C ₁ Certain amino acids of preferred targets for amino acid modifications in clostridial toxin light chains.

In one embodiment, wherein the engineered clostridial toxin is BoNT/C ₁ Said engineered BoNT/C ₁ A modification comprising at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 30, 40, or all 41) amino acid selected from the group consisting of: n6, N7, N9, D12, D15, N18, N31, E32, N55, N59, N75, N120, N121, N125, D128, Q142, N145, N177, N178, Q183, E184, D208, E211, Q247, N255, N311, E335, E339, N343, N368, N386, D389, D390, N391, Q396, N405, N407, N425, E427, D442 and N448; and said one or more amino acid modifications engineer a BoNT/C ₁ To a value that is greater than an otherwise identical BoNT/C lacking said one or more amino acid modifications ₁ 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. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modifications in the BoNT/D clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is BoNT/D, said engineered BoNT/D comprises a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 30, or all 34) amino acid selected from the group consisting of: d7, N9, D12, N15, D16, N17, D53, D73, D119, E124, E139, E142, N143, Q177, Q178, N180, E184, E255, N308, D335, N336, N339, N343, N368, N386, D389, D390, N391, D397, N403, N407, E409, E416 and N443; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/D to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/D lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modifications in the light chain of BoNT/E clostridial toxins.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, said engineered BoNT/E comprises a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 30, 35, or all 37) amino acid selected from the group consisting of: n5, N8, N10, D11, N14, D15, Q27, E28, Q53, N72, Q75, D117, N118, D121, N122, Q123, N138, N169, N170, N195, Q237, ILE244, Q290, N293, N297, D312, Q344, N362, N365, D366, N370, E373, N378, N379, N383, N390, and T397; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/E lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, said engineered BoNT/E comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, or 20) amino acid selected from the group consisting of: n5, N8, N10, D11, N14, D15, Q27, E28, N72, Q75, N118, D121, N122, Q123, N138, Q237, Q290, N297, N362, N365, D366, N378 and N379; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/E lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, said engineered BoNT/E comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, or all 19) amino acid selected from the group consisting of: n5, N8, N10, D11, N14, D15, N72, Q75, N118, N122, Q123, N138, Q237, Q290, Q297, N362, D366, N378 and N379; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/E lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, said engineered BoNT/E comprises a modification of at least one (e.g., at least 1, 2,3, 4, 5, or all 6) amino acid selected from the group consisting of: n8, N10, Q75, Q123, N138, and Q237; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/E lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is BoNT/E, said engineered BoNT/E comprises a modification of at least one (e.g., at least 1, 2, or all 3) amino acid selected from the group consisting of: q123, N138, and Q237; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/E lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modifications in the toxin light chain of BoNT/F clostridial toxins.

In one embodiment, wherein the engineered clostridial toxin is BoNT/F, said engineered BoNT/F comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 30, or all 35) amino acid selected from the group consisting of: n6, N9, N11, D12, N15, D16, D17, E28, D55, D60, D74, N76, E105, E121, N126, E127, N144, D185, N211, Q252, N305, E310, D312, N314, N329, D331, N379, D382, D383, D384, E390, N396, N400, D414 and D418; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/F to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/F lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modifications in the clostridial toxin light chain of BoNT/G.

In one embodiment, wherein the engineered clostridial toxin is BoNT/G, said engineered BoNT/G comprises a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 30, 35, or all 38) amino acid selected from the group consisting of: n4, N7, N9, N11, D12, N15, D17, E48, Q55, D57, N60, D75, D127, Q144, E148, D149, Q150, N178, E185, E208, D211, E255, D315, D332, N334, D340, E383, D387, N388, Q393, N394, E395, N403, E407, E418, E422, E426 and N443; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/G to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical BoNT/G lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The inventors have identified certain amino acids that represent preferred targets for amino acid modifications in the TeNT light chain.

In one embodiment, wherein the engineered clostridial toxin is a TeNT comprising a modification of at least one (e.g., at least 1, 2,3, 4, 5, 10, 15, 20, 30, 35, or all 36) amino acid selected from the group consisting of: n6, N7, N15, N16, D17, D31, E51, E57, N60, N76, N101, D126, D143, N167, D179, N180, E251, Q257, N313, N316, D318, D335, N337, Q339, N368, N387, D390, D391, N395, D396, E403, D406, E410, N421, D427 and E450; and the one or more amino acid modifications increase the isoelectric point (pI) of the engineered TeNT to a value that is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of an otherwise identical TeNT lacking the one or more amino acid modifications. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid with an arginine residue.

The present invention is applicable to many different types of clostridial toxins. Thus, in the context of the present invention, the term "clostridial toxin" includes the toxin produced by Clostridium botulinum (Clostridium botulinum neurotoxin serotype A, B, C ₁ D, E, F and G), Clostridium tetani (tetanus neurotoxin), Clostridium butyricum (Clostridium botulinum neurotoxin serotype E) and Clostridium pasteurianum (Clostridium botulinum neurotoxin serotype F) as well as modified clostridial toxins or toxins prepared from derivatives of any of the foregoing. The term "clostridial toxin" also includes clostridium botulinum neurotoxin serotype H.

Botulinum neurotoxin (BoNT) is prepared by clostridium botulinum in the form of a large protein complex consisting of BoNT itself complexed with a plurality of accessory proteins. There are currently 8 different classes of botulinum neurotoxins, namely: clostridium botulinum neurotoxin serotype A, B, C ₁ D, E, F, G and H, all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and this classification by serotype correlates with the percentage of sequence identity at the amino acid level. Based on the percentage of sequence identity of the amino acids, furtherThe given serotype BoNT proteins are divided into different subtypes.

BoNTs are absorbed in the gastrointestinal tract and, after entering the systemic circulation, bind to the prominent pre-membrane of cholinergic nerve terminals and prevent their release of the neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptic vesicle protein/vesicle-associated membrane protein (VAMP); BoNT/C ₁ BoNT/A and BoNT/E cleave synaptosomal associated 25kDa proteins (SNAP-25); and BoNT/C ₁ Cleaving the syntaxin.

Tetanus toxoid is prepared from clostridium tetani as a single serotype. Clostridium butyricum produces BoNT/E, and Clostridium pasteurianum produces BoNT/F.

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

The modified clostridial toxin can be in the amino acid sequence of the heavy chain (e.g., modified H) _C Domain), wherein the modified heavy chain binds to a target neural cell with higher or lower affinity than the native (unmodified) clostridial toxin. At H _C Such modifications in the domain can include modifications H _C Residues in the ganglioside binding site or in the protein (SV2 or synaptotagmin) binding site of the domain, which modification alters the ganglioside receptor and/or protein receptor binding to the target nerve cellIn combination with (c). Examples of such modified clostridial toxins are described in WO 2006/027207 and WO 2006/114308, both of which are incorporated herein by reference in their entirety.

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

The modified clostridial toxin can comprise one or more modifications that increase or decrease the biological activity and/or biological persistence of the modified clostridial toxin. For example, the modified clostridial toxin can comprise a leucine or tyrosine-based motif, wherein the motif increases or decreases the biological activity and/or biological persistence of the modified clostridial toxin. Suitable leucine-based motifs include xDxxxLL, xexxll, xexxil and xexxlm (where x is any amino acid). Suitable tyrosine-based motifs include Y-x-x-Hy (where Hy is a hydrophobic amino acid). Examples of modified clostridial toxins comprising leucine-and tyrosine-based motifs are described in WO 2002/08268, the entire contents of which are incorporated herein by reference.

The term "clostridial toxin" is intended to include hybrid and chimeric clostridial toxins. The hybrid clostridial toxin comprises at least a portion of a light chain from one clostridial toxin or subtype thereof and at least a portion of a heavy chain from another clostridial toxin or clostridial toxin subtype. In one embodiment, the hybrid clostridial toxin can comprise a complete light chain from one clostridial toxin subtype and a heavy chain from another clostridial toxin subtype. In another embodiment, the chimeric clostridial toxin can comprise a portion (e.g., a binding domain) of a heavy chain of one clostridial toxin subtype, and another portion of the heavy chain from another clostridial toxin subtype. Similarly or alternatively, the therapeutic element may comprise a light chain portion from a different clostridial toxin. Such hybrid or chimeric clostridial toxins are useful, for example, as delivery means to deliver the therapeutic benefit of such clostridial toxins to patients that are immune resistant to a given clostridial toxin subtype, to patients that may have a lower receptor concentration than the average given clostridial toxin heavy chain binding domain, or to patients that may have protease-resistant variants of membrane or vesicle toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial toxins are described in US 8,071,110, the disclosure of which is incorporated herein by reference in its entirety. Thus, in one embodiment, the engineered clostridial toxin of the invention is an engineered hybrid clostridial toxin or an engineered chimeric clostridial toxin.

The term "clostridial toxin" is intended to include retargeted clostridial toxins. In a retargeted clostridial toxin, the clostridial toxin is modified to include an exogenous ligand known as a Targeting Moiety (TM). The TM is selected to provide binding specificity to a desired target cell, and as part of the re-targeting process, the natural binding moiety of the clostridial toxin (e.g., H) _C Domain or H _CC Domains) may be removed. Retargeting techniques are described, for example, in: EP-B-0689459; WO 1994/021300; EP-B-0939818; US 6,461,617; US 7,192,596; WO 1998/007864; EP-B-0826051; US 5,989,545; US 6,395,513; US 6,962,703; WO 1996/033273; EP-B-0996468; US 7,052,702; WO 1999/017806; EP-B-1107794; US 6,632,440; WO 2000/010598; WO 2001/21213; WO 2006/059093; WO 2000/62814; WO 2000/04926; WO 1993/15766; WO 2000/61192; and WO 1999/58571, the entire contents of which are incorporated herein by reference. Thus, in one embodiment, the engineered clostridial toxins of the invention are engineered retargeted clostridial toxins.

The invention also includes clostridial toxins having a non-native protease cleavage site. In such clostridial toxins, the native protease cleavage site (also referred to as the activation site, as described above) is modified or replaced by a protease cleavage site that is not native to the clostridial toxin (i.e., an exogenous cleavage site). Such sites would require exogenous proteases for cleavage, which allows for improved control over the timing and location of the cleavage event. Non-native protease cleavage sites that can be used in clostridial toxins include:

additional protease cleavage sites include recognition sequences that are cleaved by non-cytotoxic proteases (e.g., by the light chain of clostridial neurotoxins). These include SNARE (e.g., SNAP-25, syntaxin, VAMP) protein recognition sequences that are cleaved by a non-cytotoxic protease (e.g., the light chain of clostridial neurotoxin). Clostridial toxins comprising a non-native protease cleavage site are described in US 7,132,259, EP 1206554-B2 and US 2007/0166332, which are incorporated herein by reference in their entirety. The term protease cleavage site also includes inteins, which are self-cleaving sequences. The self-splicing reaction can be controlled, for example, by varying the concentration of reducing agent present.

The invention also includes clostridial toxins comprising a "destructive cleavage site". In the clostridial toxins, a non-native protease cleavage site is incorporated into the clostridial toxin at a selected position such that cleavage at the site will reduce clostridial toxin activity or inactivation. In the event that the clostridial toxin migrates to a non-targeted location following administration, the destructive protease cleavage site may be susceptible to local protease cleavage. Suitable non-native protease cleavage sites include those described above. Clostridial toxins comprising a destructive cleavage site are described in WO 2010/094905 and WO 2002/044199, both of which are incorporated herein by reference in their entirety.

Engineered clostridial toxins of the invention, particularly the light chain components thereof, can be pegylated-this can help increase stability, e.g., duration of action of the light chain component. When the light chain contains BoNT/A, B or C ₁ With proteases, pegylation is particularly preferred. Pegylation preferably involves adding PEG to the N-terminus of the light chain component. For example, the N-terminus of the light chain may be extended by one or more amino acid (e.g., cysteine) residues, which may be the same or different. One or more of said amino groupsThe acid residue may have its own PEG molecule attached (e.g., covalently attached) thereto. Examples of such techniques are described in WO2007/104567, the entire contents of which are incorporated herein by reference.

The engineered clostridial toxins of the present invention can be devoid of complex proteins that are present in naturally occurring clostridial toxin complexes.

Engineered clostridial toxins of the invention can also comprise a limited number of non-standard amino acids. Thus, in addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α -methylserine) can be substituted for the amino acid residues of the engineered clostridial toxins of the present invention. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and unnatural amino acids can be used to replace amino acid residues of clostridial polypeptides. Engineered clostridial toxins of the present invention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2, 4-methylene-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, paconitine, tertiaryleucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used in which nonsense mutations are inhibited using the chemically aminoacylated inhibitor tRNAs. Methods for synthesizing amino acids and aminoacylating trnas are known in the art. The transcription and translation of plasmids containing nonsense mutations was performed in a cell-free system containing E.coli S30 extract and commercially available enzymes and other reagents. The protein was purified by chromatography. See, e.g., Robertson et al,J.Am.Chem.Soc.1132722.1991; the process of Ellman et al (r),Methods Enzymol.202301,1991; chung et al, in a human,Science 259806. sup. sodium chloride 9, 1993; and Chung et al, and,Proc.Natl.Acad.Sci.USA 90:10145-9,1993). In the second approach, translation was performed in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated repressor tRNAs (Turcati et al,J.Biol.Chem.271:19991-8,1996). In a third method, E.coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). A non-naturally occurring amino acid is incorporated into a polypeptide in place of its natural counterpart. See, e.g., Koide et al,Biochem.33:7470-6,1994。

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

In another aspect, the invention provides a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding an engineered clostridial toxin as described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator.

In a preferred embodiment, the vector has a promoter selected from the group consisting of:

in another preferred embodiment, the vector has a promoter selected from the group consisting of:

any suitable method known in the art may be used to prepare the nucleic acid molecules of the invention. Thus, chemical synthesis techniques can be used to prepare nucleic acid molecules. Alternatively, the nucleic acid molecules of the invention can be prepared using molecular biology techniques.

The DNA construct of the present invention is preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

The above-described nucleic acid sequence information is optionally modified for codon bias depending on the ultimate host cell (e.g., E.coli) expression system to be employed.

In one embodiment, the nucleic acid sequence encoding an engineered clostridial toxin as described above is a nucleic acid sequence that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2. 4 and 6 have at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, or 99%) sequence identity. In one embodiment, the nucleic acid sequence is identical to a sequence selected from SEQ ID NOs: 2. 4 and 6 have at least 90% sequence identity.

The invention also provides polypeptides encoded by the nucleic acid sequences as described above. Accordingly, in one aspect, the invention provides polypeptides comprising an amino acid sequence that hybridizes to a sequence selected from the group consisting of SEQ id nos: 1. 3 and 5 have at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, or 99%) sequence identity. In one embodiment, the amino acid sequence is identical to a sequence selected from SEQ ID NOs: 1. 3 and 5 have at least 90% sequence identity.

In one embodiment, the engineered clostridial toxin of the invention is an engineered BoNT/a as described above, and said engineered BoNT/a comprises (or consists of) an amino acid sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1. 3 and 5 have at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) sequence identity.

In one embodiment, the engineered clostridial toxin of the invention is an engineered BoNT/a as described above, and said engineered BoNT/a comprises the amino acid sequence of SEQ ID NO: 1. 3 or 5 (or consists thereof).

In one aspect, the invention provides a polypeptide comprising (or consisting of) the amino acid sequence of SEQ ID NO 1, 3 or 5.

In one aspect, the invention provides a nucleic acid encoding an engineered clostridial toxin as described above, wherein the nucleic acid comprises a nucleic acid sequence that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2. 4 and 6 have at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) sequence identity. In one embodiment, the nucleic acid comprises SEQ ID NO: 2. 4 or 6 (or consists thereof).

In one aspect, the invention provides a nucleic acid comprising SEQ ID NO: 2. 4 or 6 (or consists thereof).

In one embodiment, the engineered clostridial toxin of the invention is an engineered BoNT/E as described above, and said engineered BoNT/E comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 7 have at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) sequence identity.

In one aspect, the invention provides a nucleic acid encoding an engineered clostridial toxin as described above, wherein the nucleic acid comprises a nucleic acid sequence that hybridizes to SEQ ID NO: 8 have at least 70% (e.g., at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) sequence identity.

The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore,% identity can be calculated as: the number of identical nucleotides/amino acids divided by the total number of nucleotides/amino acids, multiplied by 100. Calculating percent sequence identity can also take into account the number of gaps that need to be introduced to optimize alignment of two or more sequences, as well as the length of each gap. Sequence comparisons between two or more sequences and determination of percent identity can be performed using specific mathematical algorithms (e.g., BLAST) that are familiar to the skilled artisan.

In one aspect, the invention provides a method of preparing a single-chain engineered clostridial toxin protein having a light chain and a heavy chain, the method comprising expressing a nucleic acid in a suitable host cell (the nucleic acid being as described above), lysing the host cell to provide a host cell homogenate comprising the single-chain engineered clostridial toxin protein, and isolating the single-chain engineered clostridial toxin protein.

In another aspect, the invention provides a method of activating an engineered clostridial toxin, said method comprising providing a single-chain engineered clostridial toxin protein obtainable by the method of making a single-chain engineered clostridial toxin protein 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, thereby converting the polypeptide into a two-chain polypeptide, wherein the light chain and the heavy chain are linked together by a disulfide bond.

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

In a related aspect, the invention provides an engineered clostridial toxin as described above for use in the prevention or treatment of a disease or condition selected from: strabismus, blepharospasm, torticollis, dystonia (e.g., spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, dystonia of the extremities, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic treatment (cosmetology) applications benefiting from cell/muscle disability (down-regulation or inactivation via SNARE), neuromuscular or ocular motility disorders (e.g., commonality strabismus, vertical strabismus, lateral rectus paralysis, nystagmus, thyroid dysfunction myopathy), finger spasms, blepharospasm, bruxism, hepatolenticular degeneration, tremor, tics, segmental myoclonus, spasms, spasticity caused by chronic multiple sclerosis, spasticity leading to abnormal bladder control, male imagery, back spasticity, quadriceps stiff pain, Tension headache, levator pelvic syndrome, spina bifida, tardive dyskinesia, parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anal spasm, limb spasticity, convulsions, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, muscle pain (e.g., from muscle spasm), headache (e.g., tension headache), frontal wrinkles, skin wrinkles, cancer, uterine disorder, genitourinary-neurological disorder, chronic neurogenic inflammation, and smooth muscle disorder.

In use, the invention uses a pharmaceutical composition comprising an engineered clostridial toxin and at least one component selected from the group consisting of: a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.

The engineered clostridial toxins of the invention can be formulated for oral, parenteral, continuous infusion, inhalation, or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or in the form of dry powders which are dissolved or suspended in a suitable carrier before use.

In the case of engineered clostridial toxins that are to be delivered topically, the engineered clostridial toxins can be formulated as a cream (e.g., for topical application), or for subcutaneous injection.

Topical delivery means may include an aerosol or other spray (e.g. a nebulizer). In this aspect, the aerosol formulation of engineered clostridial toxin can be delivered to the lung and/or other nasal and/or bronchial or airway passages.

Engineered clostridial toxins of the invention can be administered to a patient by intrathecal or epidural injection into the spine at the level of the spinal segment involved in innervation of the affected organs.

Preferred routes of administration are via laparoscopy and/or localised, in particular intramuscular injection.

Dosage ranges for administering the engineered clostridial toxins of the invention are those that produce the desired therapeutic effect. It will be understood that the dosage range required will depend upon the precise nature of the engineered clostridial toxin or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, degree or severity of the patient's condition, contraindications (if any), and the judgment of the attending physician. These variations in dosage levels can be adjusted using standard empirical approaches for optimization.

Suitable daily dosages (per kg body weight of the patient) are in the range of 0.0001-1ng/kg, preferably 0.0001-0.5ng/kg, more preferably 0.002-0.5ng/kg, and especially preferably 0.004-0.5 ng/kg. The unit dose can vary from less than 1 picogram to 30ng, but will typically be in the region of 0.01 to 1 ng/dose, and may be administered daily or preferably less frequently, for example weekly or every 6 months.

A particularly preferred dosing regimen is based on 0.05ng of engineered clostridial toxin as a1 x dose. In this regard, preferred dosages are in the range of 1X-100X (i.e., 0.05-5 ng).

Fluid dosage forms are typically prepared using engineered clostridial toxins and pyrogen-free sterile carriers. Depending on the vehicle and concentration used, the engineered clostridial toxin can be dissolved or suspended in the vehicle. In preparing the solution, the engineered clostridial toxin can be dissolved in a carrier, made isotonic by the addition of sodium chloride if necessary, and sterilized by filtration through a sterile filter using aseptic techniques, then filled into suitable sterile vials or ampoules and sealed. Alternatively, if solution stability is sufficient, the solution in the sealed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and or local anesthetics can be dissolved in the vehicle.

Dry powders (which are dissolved or suspended in a suitable carrier before use) may be prepared by filling pre-sterilized components in sterile containers using aseptic techniques in the sterile field. Alternatively, the components may be dissolved in a suitable container using aseptic techniques in a sterile field. The product is then freeze dried and the container is aseptically sealed.

Parenteral suspensions suitable for intramuscular, subcutaneous or intradermal injection are prepared in substantially the same manner except that the sterile components are suspended rather than dissolved in a sterile vehicle and sterilization cannot be accomplished by filtration. The components may be separated in a sterile state or, alternatively, may be sterilized after separation, for example by gamma irradiation.

Advantageously, a suspending agent such as polyvinylpyrrolidone is included in the composition to facilitate uniform distribution of the components.

Administration according to the present invention may utilize a variety of delivery techniques, including microparticle encapsulation, viral delivery systems, or high pressure aerosol impingement.

Drawings

FIG. 1 shows a schematic view of a

CatH _N V1 (FIG. 1A), CatH _N V2 (FIG. 1B) and CatH _N V3 (FIG. 1C).

FIG. 2

For CatH _N Percent of SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) of _, v 1. Rat embryonic spinal cord neurons were cultured for 3 weeks and with CatH _N A. about.v 1 for 24 hours, followed by Western blotting using SNAP-25 specific antibody. Data are mean ± SEM from three independent experiments (in triplicate).

FIG. 3

BoNT/A and CatH in mouse phrenic nerve hemiphrenic assay (mPTHHD) _N Efficacy of _V1 (t) ₅₀ ). Data points are individual hemiphrenic preparations and mean ± SEM. CatH _N V1 is statistically significantly slower than the reference protein BoNT/A (List Biological laboratories). One-way ANOVA and Dunnett's multiple comparison test. P is<0.01，***p<0.001，****p<0.0001 (one-way ANOVA and Dunnett's multiple comparison test).

FIG. 4

For CatH _N Percent of SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) of _, v 2. Rat embryonic spinal cord neurons were cultured for 3 weeks and incubated with CatH _N Treatment with _v2 for 24 hours, followed by Western blotting with SNAP-25 specific antibody. Data are mean ± SEM from three independent experiments (in triplicate).

FIG. 5

In the mouse diaphragmBoNT/A and CatH in the lateralized neural diaphragm assay (mPNHD) _N Efficacy of _V2 (t) ₅₀ ). Data points are single laterospinal preparations and mean ± SEM. CatH _N V2 is statistically equivalent to the reference protein BoNT/A (List Biological laboratories). One-way ANOVA and Dunnett's multiple comparison test. P is<0.01，***p<0.001，****p<0.0001 (one-way ANOVA and Dunnett multiple comparison test).

FIG. 6

For CatH _N Percent of SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) of _, v 3. Rat embryonic spinal cord neurons were cultured for 3 weeks and incubated with CatH _N Treatment with _v3 for 24 hours, followed by Western blotting with SNAP-25 specific antibody. Data are mean ± SEM from three independent experiments (in triplicate).

FIG. 7

BoNT/A and CatH in mouse phrenic nerve hemiphrenic assay (mPTHHD) _N Efficacy of _V3 (t) ₅₀ ). Data points are individual hemiphrenic preparations and mean ± SEM. CatH _N V3 is statistically significantly slower than the reference protein BoNT/A (List Biological laboratories). One-way ANOVA and Dunnett's multiple comparison test. P<0.01，***p<0.001，****p<0.0001 (one-way ANOVA and Dunnett multiple comparison test).

FIG. 8

Isoelectric focusing analysis. All three CatH compared to unmodified BoNT/A _N The constructs had increased pI observed.

FIG. 9

SDS-PAGE purification of CatLC constructs.

FIG. 10 shows a schematic view of a

Catalytic activity of CatLC compared to BoNT/E LC reference, using pEC obtained in BoTest A/E BoNT assay kit (BioSental Cat # A1004), according to the manufacturer's instructions ₅₀ The value is obtained. Data show mean ± sd of one independent experiment (in triplicate).

Sequence of

SEQ ID NO: 1. engineered BoNT/A, "CatH _N V1 ", amino acid sequence.

SEQ ID NO: 2. engineered BoNT/A, "CatH _N V1 ", nucleic acid sequence.

SEQ ID NO: 3. engineered BoNT/A, "CatH _N V2 ", amino acid sequence.

SEQ ID NO: 4. engineered BoNT/A, "CatH _N V2 ", nucleic acid sequence.

The amino acid sequence of SEQ ID NO: 5. engineered BoNT/A, "CatH _N V3 ", amino acid sequence.

SEQ ID NO: 6. engineered BoNT/A, "CatH _N V3 ", nucleic acid sequence.

The amino acid sequence of SEQ ID NO: 7. engineered BoNT/E light chain, "CatLC", amino acid sequence.

SEQ ID NO: 8. engineered BoNT/E light chain, "CatLC", nucleic acid sequence.

SEQ ID NO: 1. engineered BoNT/A, "CatH _N V1 ", amino acid sequence.

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKRRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMRYKRRFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSRDRPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 2. engineered BoNT/A, "CatH _N V1 ", nucleic acid sequence.

ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAcgtCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCgtTACAAAcgtcgTTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCcgtGACcgtCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG

SEQ ID NO: 3. engineered BoNT/A, "CatH _N V2 ", amino acid sequence.

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLKKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATKAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIAKKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNKIKFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLKGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

The amino acid sequence of SEQ ID NO: 4. engineered BoNT/A, "CatH _N V2 ", nucleic acid sequence.

ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAAgAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTaAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAAgAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAAgATTAAgTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGAagGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG

SEQ ID NO: 5. engineered BoNT/A, "CatH _N V3 ", amino acid sequence.

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLKPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPKLGTFALVSYKANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYKEKEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 6. engineered BoNT/A, "CatH _N V3 ", nucleic acid sequence.

ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCgAtACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAAaCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTTCATTCCGGAGATTGCGATCCCGaaGTTGGGTACCTTCGCGCTGGTGTCCTACAagGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACAaGGAAaAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG

SEQ ID NO: 7. engineered BoNT/E light chain, "CatLC", amino acid sequence.

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSMPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNKFHIGDASAVEIKFSKGSQHILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSINEFIQDPALTLMHELIHSLHGLYGAKG worker TTTCIITQQKNPLITNRKGINIEEFLTFGGNDLNIITVAQYNDIYTNLLNDYRKIASKLSKVQVSNPQLNPYKDIFQEKYGLDKDASGIYSVNINKFDDILKKLYSFTEFDLATKFQVKCRETYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIIKPITGRGLVKKIIRFAVDKLAAALEHHHHHH

SEQ ID NO: 8. engineered BoNT/E light chain, "CatLC", amino acid sequence.

ATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTCGGGATCGAGGGAAGGATTTCAGAATTCGGATCCATGCCAAAAATCAACAGCTTTAATTACAATGACCCTGTAAACGATCGTACCATCCTATACATAAAGCCGGGTGGGTGTCAAGAGTTCTACAAATCTTTCAATATTATGAAGAATATATGGATTATACCTGAGCGTAACGTTATTGGTACGACACCGCAAGATTTTCATCCACCTACTTCGTTGAAGAACGGTGACTCTTCCTATTACGACCCCAATTATCTCCAGTCGGATGAAGAGAAGGACAGATTCCTTAAAATAGTAACCAAAATCTTTAACAGGATTAATAACAATCTATCCGGAGGTATTTTGCTTGAAGAGCTTAGTAAAGCTAATCCTTACCTAGGTAACGATAATACACCAGACAACAAGTTTCATATAGGCGATGCATCCGCCGTGGAAATCAAATTTAGCAAGGGATCACAGCATATTCTCTTGCCCAACGTTATTATAATGGGGGCGGAACCAGATTTATTTGAAACAAATTCGAGTAATATTAGCCTGAGAAATAACTATATGCCGTCAAACCATGGGTTCGGTAGCATAGCGATCGTTACTTTTTCTCCCGAATACAGTTTTCGCTTCAATGATAATAGTATAAATGAGTTTATCCAAGACCCCGCACTCACGCTTATGCACGAACTCATACACTCTTTACACGGCCTGTATGGCGCTAAGGGGATAACCACTACGTGTATCATTACTCAGCAAAAGAACCCATTGATAACGAACAGGAAGGGCATTAACATCGAGGAATTTCTTACATTTGGAGGCAACGATCTGAACATTATAACTGTCGCACAGTACAATGACATCTATACCAACTTACTAAATGATTATAGAAAAATCGCTTCTAAGTTATCCAAGGTTCAAGTCTCAAACCCTCAACTGAATCCGTATAAGGACATATTCCAAGAGAAATATGGATTAGACAAAGACGCGTCAGGAATCTATTCGGTAAACATTAACAAATTCGACGATATTTTGAAGAAACTTTACAGCTTCACGGAGTTCGACTTGGCCACCAAATTCCAGGTCAAATGCCGAGAGACATACATCGGACAGTATAAGTATTTCAAGCTGTCGAATCTCCTGAATGATTCCATATACAACATTAGTGAGGGTTACAATATAAATAACCTAAAGGTGAATTTCCGAGGCCAAAACGCCAACCTAAATCCGCGCATCATTAAACCCATCACAGGACGGGGGTTAGTGAAGAAAATAATCCGGTTTGCGGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCAC

Examples

The following examples are intended to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined in the claims in any way.

Example 1

Preferred clostridial toxin amino acids for modification are identified.

Amino acids identified as suitable candidates for modification (mutation sites) are selected using a variety of different criteria.

Position of residue within BoNT molecule (H) _N Inner, excluding band regions);

2. the location of secondary/tertiary structures;

3. the type of residue;

4. degree of surface exposure.

For selection, acidic, neutral, polar and hydrophobic residues are considered.

Exposed residues were determined using AreaIMol in the CCP4 kit. Each structure was analyzed by AreaIMol and the exposed residues were identified as having a sum greater than 55.

Identification of H for each subtype and TeNT Using the Secondary Structure Allocation program (Stride Web Interface) _N Secondary structure within. Exclusion of partitioning into alpha-helices, beta-strands or 3 from selection ₁₀ -the area of the helix.

Sequences used

Accession number:

BoNT/A：P10845

BoNT/B：P10844

BoNT/C ₁ ：P18640

BoNT/D：P19321

BoNT/E：Q00496

BoNT/F：YP_001390123

BoNT/G：Q60393

structural data source

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

B was performed using LOOPP and the following sequences, respectively _o NT/C ₁ 、B _o NT/D、B _o Homology modeling of NT/F and BoNT/G: p18640, P19321, YP _001390123, and Q60393.

Clostridial toxin H _N Preferred clostridial toxin amino acid residues for modification in the domain:

BoNT/A：

d474, N476, D484, N486, I487, E488, a489, a490, E491, D546, E558, E560, H561, I566, L568, N570, S571, L577, N578, a597, E599, a601, E620, V621, T623, D625, T631, N645, L647, D650, D651, I668, E670, a672, V675, S683, I685, a686, N687, N752, Q753, T755, E757, E758, N760, N761, I762, N763, D825, I831, G832, T847, D848, and D858

BoNT/B：

V443, G444, D453, S468, D533, E534, N535, T545, L548, D549, I550, D552, S557, L564, S566, N582, V584, N609, L619, N632, E633, G637, a646, I655, E657, V662, E669, S670, I672, D673, N739, I740, N748, N750, I818, G819, T834, I842, N845, and S858

BoNT/C ₁ ：

L451, D452, C453, E455, V472, T474, D475, L478, N483, E484, E485, E487, I489, L555, S556, D557, N558, E560, D561, E569, N574, S575, T584, G592, Q594, G596, D617, N640, S641, V642, G645, N646, E661, E665, T667, A670, S678, V680, Q68L, E682, S750, G84751, S759, Q760, V826, G827, N842, T843, N7, and N853

BoNT/D：

Q469, E470, E473, N474, D479, E480, N482, V483, Q484, N485, S487, D488, S552, N553, N554, V555, E556, N557, I558, L560, T562, S563, V564, G569, S571, N572, G588, Q590, T614, D616, S619, S622, N636, S637, L639, G641, N642, E657, E818, T663, a666, V669, S674, I676, Q677, E678, S746, G747, D749, E751, N752, I753, Q756, N818, V822, G823, E837, N838, T839, N843, N849, and N850

BoNT/E：

D474, N476, E479, E480, D484, N486, I487, E488, a489, a490, E491, E492, L496, D497, Q500, Q501, L504, N507, D509, N510, N514, S516, E518, Q527, L530, N533, I534, E535, N539, Y548, I566, L568, D589, a597, E599, a601, L604, Y612, E620, N645, L647, Y648, D651, E737, E741, Y803, Y824, D825, G831, I831, G832, and D835

BoNT/F：

N463, E464, N468, T469, D474, D475, T476, T477, N478, N482, N485, N495, I499, Q501, I502, Q505, T506, N508, T509, V511, D513, D521, S522, S526, E527, I528, E529, V534, D535, L536, E549, G550, T552, N553, S558, E566, E567, S568, V, H587, Q608, D613, a 586, D617, S619, N630, N633, N639, E654, V656, E658, L660, T663, L665, V666, S671, I673, G674, S675, S676, E677, N678, T746, N852, L756, E754, E751, N759, N75821, N848, N821, S841, S856, S841, S845, S841, S847, S841, S849, S855, N849, N758, S851, L849, S758, L849, N855, N758, S851, S855, S779, S855, S779, S855, N855, S779, S855, S779, S855, S779, S855, S779, S77s 779, S855, S77s 855, S779, S77s 779, S855, S779, S855, S77s 855, S77s 855, S779, S77s 855, S77s 779, S77s S779, N855, S779, S77s 549, S77s 779, S77s 779, S855, S779, S77s 855, S779, S549, S779, S77s 779, S779, N I S549, S02, S

BoNT/G：

N480, Q482, N483, N484, T485, E487, D540, N562, N570, N571, N572, T588, V589, T615, D621, N637, E638, E642, N643, I660, E662, I667, E674, S675, V677, G678, N679, S747, N755, D757, L823, D839, I841, D844, S846, and L847

TeNT：

A457, S458, L459, D461, L462, E486, E487, Q490, D491, N497, N504, D557, T571, T572, L573, Q574, N580, S581, N588, S589, T590, S598, Q605, G606, Q608, T631, I633, S640, Q655, E658, G659, N660, E675, I677, E679, T681, V684, A691, E692, S694, T695, Q696, A772, D773, E774, S862, N866, L867 and D866

Preferred clostridial toxin amino acid residues for modification in the BoNT/E light chain:

n5, N8, N10, D11, N14, D15, Q27, E28, N72, Q75, N118, D121, N122, Q123, N138, Q237, Q290, N297, N362, N365, D366, N378, and N379.

Example 2

Design of engineered BoNT/A molecules

Three different examples of engineered BoNT/a molecules according to the present invention were prepared.

Using the method described in example 1, a total of 55 residues were identified as BoNT/A H _N Candidates for mutations in the domain. The suitability of the residues was further evaluated by visual inspection of BoNT/a crystal structure, listing 11 preferred candidates (N476, N763, N687, E599, I831, N761, N578, V675, I685, T755, E757). The other 6 residues were selected based on functional data showing that these residues are suitable for mutation without adversely affecting protein function. 4 of these residues are in the candidate list (L647, D650, D651, T847) and 2 are not (S564, I849). 11 residues in the candidate list plus 6 residues in the functional data, a total of 17 residues were selected for mutation.

From the 17 residues selected, 3 constructs were made: CatH _N _v1、CatH _N _ v2 and CatH _N _v3。CatH _N Mutations of the constructs are shown in table 3 below:

table 3: CatH with mutations listed _N Constructs and calculated pI.

[ Delta pI-Change in isoelectric Point ]

CatH _N _v1、CatH _N _ v2 and CatH _N Purification of iv 3 is shown in FIGS. 1A, 1B and 1C, respectively.

Example 3

Cloning, expression and purification

The DNA construct encoding the engineered BoNT/A molecule described in example 2 was synthesized, cloned into the pJ401 expression vector and then transformed into BL21(DE3) E.coli. This allows soluble overexpression of the recombinantly engineered BoNT/A molecule in E.coli.

The recombinant engineered BoNTs were purified from e.coli lysates using conventional chromatographic techniques. An initial purification step using a cation exchange resin is applied, followed by an intermediate purification step using a hydrophobic interaction resin. The recombinantly engineered BoNT single strands are then cleaved proteolytically to yield activated double-stranded engineered BoNT. The remaining contaminants are then removed using a final purification step.

Example 4

Characterization of purified engineered BoNTs

The engineered BoNTs described in example 2 above were characterized experimentally.

Rat embryonic spinal cord neurons (eSCN) were used to assess the ability of engineered BoNTs to enter neurons and cleave SNAP-25 (the target of BoNT/A). The efficacy of engineered BoNTs was further evaluated using the mouse phrenic nerve hemiphrenic assay (mPNHD).

CatH _N _v1：

The first set of mutations added were substitutions for arginine:

S564R、L647R、D650R、D651R、T847R、I849R

testing CatH in the SNAP-25 cleavage assay for rat embryonic spinal cord neurons (eSCN) _N The _v1 molecule, and was found to have a relationship with BoNT/A (BoNT/A)Equal efficacy (fig. 2).

Positive results were also confirmed in the mouse phrenic nerve hemiphrenic (mPNHD) assay (fig. 3).

CatH _N _v2：

The second group of mutations was lysine:

N476K、N763K、N687K、E599K、I831K、N761K

CatH was tested in the eSCN SNAP-25 cleavage assay _N An _v2 protein, and was found to retain the ability to enter cells and cleave SNAP-25. In the mPTH assay, CatH _N _ v2 is equivalent to BoNT/A (FIGS. 4 and 5).

CatH _N _v3：

The third group of mutated substitutions is lysine:

N578K、V675K、I685K、T755K、E757K

testing CatH in eSCN SNAP-25 cleavage assay _N The _v3 molecule, and was found to retain the ability to enter cells and cleave SNAP-25. Similarly, positive results were also confirmed in the mphnd assay (fig. 6 and 7).

Isoelectric focusing

All three CatH compared to unmodified BoNT/A _N The construct had an increased pI (fig. 8).

Example 5

Modifications in BoNT/E light chain

Due to the modular nature of botulinum toxin, a BoNT/E light chain construct with an N-terminal Maltose Binding Protein (MBP) tag and a C-terminal 6 histidine tag (6HT) was used as an alternative to determine BoNT/E activity upon mutation and characterization.

BoNT/E light chain constructs ("CatLC") were prepared with the mutations shown in the following table.

Table 4: constructs with the listed mutations, calculated pI and calculated Δ pI.

This construct was expressed in BL21DE3 cells and purified using affinity chromatography. Purification is shown in FIG. 9.

Evaluation of the catalytic activity of the constructs showed that the modified light chain retained the catalytic activity of the unmodified light chain (fig. 10).

Claims (6)

1. An engineered Clostridium botulinum neurotoxin A (BoNT/A) having the amino acid sequence of SEQ ID No: 1. 3 or 5.

2. A nucleic acid encoding the engineered BoNT/a of claim 1.

3. A method for preparing the engineered BoNT/a of claim 1, the protein having a light chain and a heavy chain, said method comprising expressing the nucleic acid of claim 2 in a suitable host cell, lysing the host cell to provide a host cell homogenate comprising single-chain engineered BoNT/a, and isolating the single-chain engineered BoNT/a.

4. The method of claim 3, further comprising contacting said single-chain engineered BoNT/A with a protease that cleaves said single-chain engineered BoNT/A at a recognition site located between a light chain and a heavy chain, and converting said single-chain engineered BoNT/A into a double-chain BoNT/A, wherein the light chain and the heavy chain are linked together by a disulfide bond.

5. Use of the engineered BoNT/a of claim 1 or obtained by the process of claim 4 in the manufacture of a medicament for the prevention or treatment of a disease or condition selected from: strabismus, dystonia, torticollis, neuromuscular disorders or ocular motility disorders, bruxism, hepatolenticular degeneration, tremor, tics, segmental myoclonus, spasticity caused by chronic multiple sclerosis, spasticity leading to abnormal bladder control, levator pelvic syndrome, spina bifida, tardive dyskinesia, parkinson's disease, stuttering, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, spasticity of the extremities, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, muscular pain, headache, bone tumors, uterine disorders, genitourinary disorders, chronic neurogenic inflammation, smooth muscle disorders, and skin wrinkles.

6. The use of claim 5, wherein:

the dystonia is spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, dystonia of the extremities, or dystonia of the cervix; or

The torticollis is a spastic torticollis; or

The neuromuscular disorder or ocular motility disorder is common strabismus, strabismus vertically, paralysis of the external rectus muscle, nystagmus, thyroid dysfunction myopathy; or

The muscle pain is pain from a muscle spasm or quadriceps stiffness pain; or

The skin wrinkles are frontal wrinkles; or

The spasm is blepharospasm, dactylospasm, back spasm, hemifacial spasm, or anal spasm; or the genitourinary disorder is a urogenital-neurological disorder; or

The headache is a tension headache.