JP2021104047A - Cationic neurotoxin - Google Patents

Abstract

To provide engineered clostridial toxins comprising at least one amino acid modification, and to provide methods of using such engineered clostridial toxins in medicine and therapy.SOLUTION: There is provided an engineered clostridial toxin comprising at least one amino acid modification, the at least one amino acid modification increasing 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 the at least one amino acid modification, and the at least one amino acid modification being not located in the clostridial toxin binding domain (HC domain).SELECTED DRAWING: Figure 2

Description

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

Bacteria of the genus Clostridia produce highly potent, specific protein toxins that can be toxic to the nerves and other cells to which they are delivered. Examples of such Clostridium toxins are the neurotoxin (TeNT) produced by C. tetani and the neurotoxin (BoNT) serum types A to G and C. balati (BoNT) produced by C. botulinum (botulinum). Examples include those produced by baratii) and C. butyricum.

Among the Clostridium toxins, there are some of the most powerful toxins known. _{As an example, botulinum neurotoxin has a median lethal dose (LD 50} ) value in mice ranging from 0.5 to 5 ng / kg, depending on the serotype. Both tetanus toxin and botulinum toxin act by inhibiting the functioning of affected nerves, specifically the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, whereas Clostridium tetani toxin acts in the central nervous system.

In fact, the clostridium toxin is synthesized as a single-chain polypeptide that is post-translationally modified by a cleavage event due to protein cleavage 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. This double-stranded form is the active form of the toxin. The two chains are called a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains an N-terminal translocation component (H _N domain) and a C-terminal targeting component (H _C domain). The cleavage site is located between the L chain and the translocation domain component. After _{binding of the H C} domain to its target neuron and internal translocation of the endosomally bound toxin into the cell, the H _N domain translocates the L chain across the endosome membrane into the cytosol and L The strands provide protease function (also known as non-cytotoxic proteases).

Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (eg, SNAP-25, VAMP or syntaxin) (Gerald K (2002), Cell and Molecular Biology). (4th edition) John Wiley & Sons, see Inc.). The acronym SNARE is derived from the term Soluble NSF Attachment Receptor; where NSF means N-ethylmaleimide-susceptibility factor. SNARE proteins are essential for intracellular vesicle fusion and therefore for molecular secretion by vesicle transport from the cell. Protease function is zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Thus, non-cytotoxic proteases, once delivered to the desired target cell, can inhibit cellular secretion from the target cell. The Clostridium toxin L-chain protease is a non-cytotoxic protease that cleaves the SNARE protein.

Given the ubiquity of SNARE proteins, Clostridium toxins such as botulinum toxin have been successfully used in a wide range of therapies.

As an example, we have botulinum toxin (BoNT), BoNT / A, BoNT / B, BoNT / C ₁ , for inhibiting neurotransmission in some therapeutic and cosmetic or aesthetic applications. Literature describing the use of Clostridium toxins such as BoNT / D, BoNT / E, BoNT / F and BoNT / G and tetanus neurotoxin (TeNT) (William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc.). , 2004))). For example, over-the-counter botulinum toxin products include localized spasticity, upper limb spasticity, lower limb spasticity, cervical dystonia, blepharospasm, hemifacial spasm, axillary sweating, chronic migraine, neurogenic urinary muscle overactivity, line between the eyebrows. And is currently approved for the treatment of indications involving severe hemifacial lines. In addition, Crostridium toxin therapy is used to treat neuromuscular disorders (see (US6,872,397)); to treat uterine disorders (see (US2004 / 0175399)); ulcers and gastroesophageal To treat reflux disease (see (US2004 / 0086531)); to treat dystonia (see (US6,319,505)); to treat eye disorders ((US2004 / 0234532) ); To treat eyelid spasm (see (US2004 / 0151740)); to treat strabismus (see (US2004 / 0126396)); to treat pain To (see (US6,869,610), (US6,641,820), (US6,464,986) and (US6,113,915)); to treat fibromyalgia ((US6,623,742), ( See US2004 / 0062776)); To treat lower back pain (see (US2004 / 0037852)); To treat muscle injuries (see (US6,423,319)); To treat rhinitis headaches (see (US6,838,434)); to treat tension headaches (see (US6,776,992)); to treat headaches (see (US6,776,992)) , 458,365)); To reduce migraine pain (see (US5,714,469)); To treat cardiovascular disease (see (US6,767,544)); To treat neurological disorders such as Parkinson's disease (see (US6,620,415), (US6,306,403)); to treat neuropsychiatric disorders ((US2004 / 0180061), (US2003 / 0211121)) See); To treat endocrine disorders (see (US6,827,931)); To treat thyroid disorders (see (US6,740,321)); Sweat glands affected by cholinergic action To treat disorders (see (US6,683,049)); To treat diabetes (see (US6,337,075), (US6,416,765)); To treat pancreatic disorders (see (US6,437,075), (US6,416,765)) (See US6,261,572), (US6,143,306)); Treat cancers such as bone tumors For (see (US6,565,870), (US6,368,605), (US6,139,845), (US2005 / 0031648)); for treating ear disorders ((US6,358,926), (US6, See 265,379)); To treat autonomic neuropathy such as gastrointestinal muscular disorders and other smooth muscle dysfunctions (see (US 5,437,291)); Skin lesions associated with cutaneous cell proliferative disorders For the treatment of (see (US5,670,484)); for the management of neuroinflammatory disorders (see (US6,063,768)); for the reduction of hair loss and stimulation of hair growth (see (US6,063,768)) (See (US6,299,893)); for treatment of downwardly bent mouth (see (US6,358,917)); to reduce appetite (see (US2004 / 40253274)) For dental treatments and procedures (see (US2004 / 0115139)); for treating neuromuscular disorders and conditions (see (US2002 / 0010138)); various disorders and conditions and associations To treat pain (see (US2004 / 0013692)); to treat conditions caused by hypersecretion of mucus such as asthma and COPD (see (WO00 / 10598)); and inflammation, It has been described to treat non-neurological conditions such as endocrine, exocrine, immunological, cardiovascular, and bone conditions (see (WO01 / 21213)). All of the above publications are incorporated herein by reference in their entirety.

The use of non-cytotoxic proteases such as Clostridium toxins (eg, BoNT and TeNT) in the therapeutic and cosmetic treatment of humans and other mammals can benefit from the properties of these toxins, a wide range of diseases. And is expected to spread to illness.

To avoid systemic neurological effects, many Clostridium toxin therapies administer the Clostridium toxin therapeutic agent directly to a given target site (such as the target tissue). The problem with administering Clostridium toxin-based therapeutics in this manner is the spread of the toxin to surrounding tissues or systemic circulation away from the site of administration. The spread of toxins away from the target tissue is believed to cause unwanted side effects that can be life-threatening in extreme cases. This can be a particular concern when using Clostridium toxin remedies (such as BoNT remedies) at high doses, concentrations and injection volumes. Adverse effects associated with this problem reported for over-the-counter BoNT / A treatments include asthenia, general weakness, diplopia, ptosis, dysphagia, dysarthria, dysarthria, urinary incontinence and dyspnea. Can be mentioned. Swallowing and dyspnea can be life-threatening, and deaths associated with the spread of toxin effects have been reported.

US6,872,397
US2004 / 0175399
US2004 / 0086531
US6,319,505
US2004 / 0234532
US2004 / 0151740
US2004 / 0126396
US6,869,610
US6,641,820
US6,464,986
US6,113,915
US6,623,742
US2004 / 0062776
US2004 / 0037852
US6,423,319
US6,838,434
US6,776,992
US6,458,365
US5,714,469
US6,767,544
US6,620,415
US6,306,403
US2004 / 0180061
US2003 / 0211121
US6,827,931
US6,740,321
US6,683,049
US6,337,075
US6,416,765
US6,261,572
US6,143,306
US6,565,870
US6,368,605
US6,139,845
US2005 / 0031648
US6,358,926
US6,265,379
US5,437,291
US5,670,484
US6,063,768
US6,299,893
US6,358,917
US2004 / 40253274
US2004 / 0115139
US2002 / 0010138
US2004 / 0013692
WO00 / 10598
WO01 / 21213

Gerald K (2002) "Cell and Molecular Biology" (4th Edition) John Wiley & Sons, Inc.
William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004)

Therefore, there is a need in the art for Clostridium toxins that have the property of increased retention in tissues at the site of administration as compared to known Clostridium toxins and therefore exhibit reduced diffusion away from the site of administration.

The present invention solves the above problems by providing a modified Clostridium toxin as specified in the claims.

In one aspect, the invention provides a modified crostridium toxin comprising at least one (eg, at least one, two or three) amino acid modifications, wherein the at least one amino acid modification is the modified crostridium. The isoelectric point (pI) of the toxin is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, at least 0.2, 0.3, 0.4, 0.5, 0.6, at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6,) than the pI of the same clotridium toxin, which lacks at least one amino acid modification described above. 0.7, 0.8, 0.9, or 1) increase in pI units higher value, at least one amino acid modification of the are not located in the Clostridial toxin binding domain (H _C domain).

In one embodiment, "not located in the Clostridium toxin binding domain (H _C ) domain" means that at least one of the amino acid modifications described above is located in the Clostridium toxin H _N domain or in the Clostridium toxin light chain. means.

In one embodiment, the invention provides a modified crostridium toxin comprising at least one (eg, at least one, two or three) amino acid modifications, wherein the at least one amino acid modification described above is a modified crostridium. The isoelectric point (pI) of the toxin is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, at least 0.2, 0.3, 0.4, 0.5, 0.6, at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6,) than the pI of the same clotridium toxin, which lacks at least one amino acid modification described above. 0.7, 0.8, 0.9, or 1) pI units increased to higher values, and at least one amino acid modification described above is located within the crostridium toxin translocation domain (H _N domain).

In another embodiment, the invention provides a modified crotridium toxin comprising at least one (eg, at least one, two or three) amino acid modifications, wherein the at least one amino acid modification is modified. The isoelectric point (pI) of the crostridium toxin is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6) than the pI of the same clostridium toxin that lacks at least one amino acid modification described above. , 0.7, 0.8, 0.9, or 1) pI units increased to higher values, and at least one amino acid modification described above is located in the crostridium toxin light chain.

In one embodiment, the at least one amino acid modification is located in the crostridium toxin light chain and the at least one amino acid modification does not introduce an E3 ligase recognition motif into the crostridium toxin light chain. Thus, in one embodiment, the light chain of the modified Clostridium toxin of the present invention does not contain an E3 ligase recognition motif.

As used above, the term "E3 ligase recognition motif" refers to the modification of the light chain that results in accelerated degradation of the neurotoxic polypeptide by the endogenous proteasome degradation pathway present in the subject to which the neurotoxin was applied. Point to. The "E3 ligase recognition" motif is the recognition and motif of the motif by the E3 ligase (also known as the E3 ubiquitin ligase; therefore, the "E3 ligase recognition motif" is also sometimes referred to as the "E3 ubiquitin ligase recognition motif"). It is a structural motif that enables the combination of. E3 ligase recognition motifs will be familiar to those skilled in the art.

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

Further examples of E3 ligase recognition motifs are 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 in which the at least one amino acid modification is located in the crostridium toxin light chain, the at least one amino acid modification does not introduce the MDM2 E3 ligase recognition motif into the crostridium toxin light chain. Thus, in one embodiment, the light chain of the modified Clostridium toxin of the present invention does not contain the MDM2 E3 ligase recognition motif.

In one embodiment in which at least one amino acid modification described above is located in the Clostridium toxin light chain, the modified Clostridium toxin does not include an amino acid modification in the N-terminal proline.

In one embodiment where the modified Clostridium toxin is BoNT / E and at least one of the amino acid modifications is located in the Clostridium toxin light chain, the modified Clostridium toxin has the following amino acid positions: Q53, N72, N378, One of N379, R394, T400 does not include substitution with lysine.

In one embodiment where the modified Clostridium toxin is BoNT / E and at least one of the amino acid modifications is located in the Clostridium toxin light chain, the modified Clostridium toxin has the following amino acid positions: Q53, N72, N378: , N379, T400, and does not include substitution with lysine.

In one embodiment where the modified Clostridium toxin is BoNT / E and at least one of the amino acid modifications is located in the Clostridium toxin light chain, the modified Clostridium toxin has the following amino acid positions: Q53, N72, N378: , N379, R394, T400, and does not include substitution with lysine.

In one embodiment where the modified Clostridium toxin is BoNT / E and at least one of the amino acid modifications is located in the Clostridium toxin light chain, the modified Clostridium toxin has the following amino acid positions: Q53, N72, N378, Any three of N379 and T400 do not include substitution with lysine.

Optionally, in one embodiment where at least one amino acid modification is located in the Clostridium toxin light chain, the modified Clostridium toxin is not BoNT / E.

The modified Clostridium toxins of the present invention do not contain any amino acid modifications located in _{the Clostridium toxin H C domain.} Thus, the modified Clostridial toxin of the present invention, at least one amino acid modification of the are not located in the Clostridial toxin H _C domain.

In one embodiment in which the at least one amino acid modification is located in the clostridium toxin light chain, the at least one amino acid modification does not involve substitution of the amino acid residue with a lysine residue.

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

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

In one embodiment where the modified Clostridium toxin is a modified Clostridium toxin as described above, at least one amino acid modification described above lacks the pI of the modified Clostridium toxin, otherwise at least one amino acid modification described above. It increases to a value at least 0.4 pI units higher than the pI of the same Clostridium toxin. In one embodiment, the at least one amino acid modification is at least 0.5 pI units higher than the pI of the modified Clostridium toxin, otherwise it lacks the at least one amino acid modification of the same Clostridium toxin. Increase to value. In one embodiment, the at least one amino acid modification is at least 0.6 pI units higher than the pI of the modified Clostridium toxin, otherwise it lacks the at least one amino acid modification of the same Clostridium toxin. Increase to value. In one embodiment, the at least one amino acid modification is at least 0.8 pI units higher than the pI of the modified Clostridium toxin, otherwise the pI of the same Clostridium toxin lacking the at least one amino acid modification. Increase to value. In one embodiment, the at least one amino acid modification is at least 1 pI unit higher than the pI of the modified Clostridium toxin, otherwise the pI of the same Clostridium toxin lacking the at least one amino acid modification. Increases to.

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

In certain embodiments, the at least one amino acid modification is at least a few more than the pI of the modified Clostridium toxin, otherwise the pI of the same Clostridium toxin lacking the at least one amino acid modification. , 4 or 5 pI units higher.

In certain embodiments, the modified Clostridium toxin comprises at least three amino acid modifications, said at least three amino acid modifications lacking the pI of the modified Clostridium toxin, otherwise at least three amino acid modifications. It increases to a value at least 0.2 pI units higher than the pI of the same Clostridium toxin.

In certain embodiments, the modified Clostridium toxin comprises at least 5 amino acid modifications, said at least 5 amino acid modifications lacking the pI of the modified Clostridium toxin, and otherwise lacking at least 5 amino acid modifications. It increases to a value at least 0.5 pI units higher than the pI of the same Clostridium toxin.

We have obtained modified Clostridium by increasing the pI of Clostridium toxin, for example by at least 0.2 pI units or 0.5 pI units or 1 pI units (by introduction of at least one amino acid modification into the Clostridium toxin protein). The toxin advantageously demonstrates the properties of increased tissue retention and reduced spread away from the site of administration, while retaining the ability of target cell binding, translocation and cleavage of the target SNARE protein (s). I found that. Therefore, the spread of Clostridium toxin from the administration site is significantly reduced compared to the same Clostridium toxin in that it lacks at least one amino acid modification.

The modified Clostridium toxins of the present invention are suitable for use in any of the above therapies and can advantageously demonstrate reduced or no side effects compared to the use of known Clostridium toxin therapeutic agents. Increased tissue retention properties of the modified Clostridium toxins of the invention also provide increased efficacy and / or duration of action compared to known Clostridium toxin therapeutics, with reduced doses (or additional adverse effects). No increased dose) can be used, thus providing additional benefits.

Thus, in one embodiment, the modified Clostridium toxins of the invention have increased potency, increased retention in tissues and / or increased duration of action as compared to the corresponding unmodified Clostridium toxins.

As discussed in more detail below, the increase in pI provided by at least one amino acid modification is that the modified Clostridium toxin of the invention lacks at least one amino acid modification described above at a given pH, etc. In this respect, it means having a net charge that is more positive than the net charge for the same Clostridium toxin. Without going to be caught up in any one theory, we believe that this increased positive charge causes the modified proteoglycan toxin of the invention to be the modified proteoglycan toxin and anionic cells at the site of administration. It is believed that favorable electrostatic interactions between extracellular components (such as cell membranes and proteoglycans with heparin sulfate) allow for longer tissue retention times at the site of administration. These improved electrostatic interactions work to reduce the diffusion of modified Clostridium toxin away from the administration site and thus improve tissue retention.

As an example, the improved tissue retention properties of the modified crotridium toxins of the present invention are: (i) higher doses to individual muscles such as the sternocleidomastoid muscle that do not spread to adjacent muscles in the neck causing dyspnea. And (ii) a higher total dose (to all muscles) in a single treatment that does not spread to the circulation and does not cause systemic effects such as dyspnea may be possible. Benefits for the patient include more effective treatment of large muscles such as the sternocleidomastoid muscle, increased chances of injecting into several different muscles between each treatment and longer effective treatment with higher dosing (more) (Long, until retreatment is needed) can be mentioned.

In one embodiment, the modified Clostridium toxins of the invention have a positive net charge in use (eg, where the modified Clostridium toxin is located in the tissue at the desired site of administration in use).

The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are made up of specific sequences of amino acids (also called amino acid residues in proteins). Each amino acid in the 20 standard sets has different side chains (or R groups), meaning that each amino acid residue in a protein exhibits different chemical properties such as charge and hydrophobicity. These properties can be affected by the surrounding chemical environment such as temperature and pH. The overall chemical characteristics of a protein depend on the sum of these various factors.

Certain amino acid residues (detailed below) have an ionizable side chain that can exhibit a charge depending on the ambient pH. Whether such side chains are charged at a given pH depends on the pKa of the relevant ionizable moiety, which is the acid dissociation constant (Ka) of a particular proton derived from a conjugate base. It is a negative logarithm.

For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with a pKa value of approximately 4.1 (the exact pKa value can vary depending on temperature, ionic strength and the microenvironment of the group that can be ionized. ). Therefore, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as the "physiological pH"). At low pH values, these side chains become protonated and lose their charge.

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

Therefore, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues (and its surface exposure) present in the protein, and on the ambient pH. Changing the ambient pH changes the overall charge on the protein. Therefore, for any protein, there is a given pH where the number of positive and negative charges is equal and the protein does not exhibit an overall net charge. This point is known as the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry that is familiar to those skilled in the art.

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

Methods of determining the pI of a protein are known in the art and well known to those of skill in the art. As an example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein (“calculated pI”). Such calculations can be performed using computer programs known in the art; as preferred examples of computer programs for calculating pI values, the Protein Calculator from Scripps Research Institute and the Compute pI / from ExPASy. MW Tool can be mentioned. Comparison of pI values between different molecules must be done using the same calculation technique / program.

Where appropriate, the calculated pI of the protein can be confirmed experimentally using the technique of isoelectric focusing (“measured pI”). This technique uses electrophoresis to separate proteins according to their pI. Isoelectric focusing is usually performed using gels with an immobilized pH gradient. When an electric field is applied, the protein moves by a pH gradient until it reaches a pH with a net charge of zero, which is the pI of the protein. The results provided by isoelectric focusing are usually relatively low resolution by their nature, and we find that the results provided by calculated pI (as described above) are more suitable for use. I believe that.

Throughout this specification, "pI" means "calculated pI" unless otherwise noted.

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

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

As described above, in one embodiment, the modified crotridium toxin of the present invention comprises at least one amino acid modification, wherein the at least one amino acid modification comprises the isoelectric point (pI) of the modified crotridium toxin. It lacks at least one of the amino acid modifications described above and otherwise increases to a value at least 0.2 pI units higher than the pI of the same crostridium toxin.

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

As described above, in one embodiment, the modified crotridium toxin of the present invention comprises at least one amino acid modification, wherein the at least one amino acid modification comprises the isoelectric point (pI) of the modified crotridium toxin. Lacking at least one of the amino acid modifications described above, it increases to a value at least 1 pI unit higher than the pI of the same crostridium toxin in other respects.

Therefore, in the context of the present invention, a 1 unit increase in pI in the context of modified BoNT / A Clostridium toxin results in an increase in pI from 6.4 to 7.4.

In one embodiment, the modified Clostridium toxin has a pI of at least 5.5.

In one embodiment, the modified Clostridium toxin has at least 6 (eg, at least 6, at least 7, at least 8 or at least 9) pI.

In one embodiment, the modified Clostridium toxin has a pI of at least 6.5.

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

In one embodiment, the modified Clostridium toxin has a pI between 6.5 and 9.5 (eg, a pI between 6.5 and 7.5).

As discussed above, the modified Clostridium toxins of the invention also provide increased efficacy and / or duration of action compared to known Clostridium toxin therapeutics, with reduced doses (or additional adverse effects). It has increased tissue retention properties that can allow no increased dose) to be used. One way these favorable properties (corresponding to an increase in therapeutic index) can be defined is in terms of the factor of safety of the modified Clostridium toxin. In this regard, the undesired effect of Clostridium toxin (caused by the diffusion of toxin away from the site of administration) is weight loss in related animal models (eg, mice in which weight loss is detected within 7 days of administration). It can be evaluated experimentally by measuring the percentage of. Conversely, the desired on-target effect of Clostridium toxin can be assessed experimentally by the Digital Abduction Score (DAS) assay, a scale of muscle paralysis. The DAS assay was performed by injecting 20 μl of Clostridium toxin into the mouse gastrocnemius / soleus complex formulated with Gelatin Phosphate Buffer, followed by Aoki's method (Aoki KR, Toxicon, Vol. 39, 1815-1820). It can be carried out by the evaluation of Digital Abduction Score using (2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response that stretches their limbs and then abducts their fingers. After injection of Clostridium toxin, fluctuating finger abduction is scored on a 5-point scale (0 = normal-4 = maximal reduction in finger abduction and limb extension).

The safety factor of Clostridium toxin was then the amount of toxin required for a 10% reduction in body weight (measured by peak effect within the first 7 days after dosing in mice) and the amount of toxin required for a DAS score of 2. Can be expressed as a ratio between. Therefore, a high factor of safety score is desired, indicating a toxin that can efficiently paralyze the target muscle with little undesired off-target effect. The modified toxins of the present invention have a higher factor of safety than the equivalent unmodified (natural) botulinum toxin.

Thus, in one embodiment, the modified clotridium toxins of the invention have a factor of safety of at least 8 (eg, at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50). Here, the factor of safety is calculated as the dose of toxin required for a -10% weight change (pg / mouse) divided by _{DAS ED 50 (pg / mouse) [ED 50} yields a DAS score of 2]. The dose required for].

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

The modified Clostridium toxin of the present invention comprises at least one amino acid modification. The at least one amino acid modification described above increases the pI of Clostridium toxin, as discussed above. In the context of the present invention, amino acid modification is modification of the amino acid sequence of Clostridium toxin. Such modifications can be achieved by replacing one amino acid in the sequence with another (ie, substituting), inserting a new amino acid into the sequence, or deleting an amino acid in the sequence. Amino acids incorporated into the amino acid sequence of a protein are also called amino acid residues.

The 20 standard amino acids found in proteins are:

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

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

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

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

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

In one embodiment, 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 Clostridium toxin amino acid sequence are replaced with different amino acid residues. The substituted amino acid residue can be one of the 20 standard amino acids as described above.

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

Amino acid insertion incorporates additional amino acid residues (those that are not normally present) into the crostridium toxin amino acid sequence, thus increasing the total number of amino acid residues in the sequence. Amino acid deletion removes amino acid residues from the Clostridium toxin amino acid sequence and thus reduces 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. As an example, amino acid modifications can be introduced by modifying the DNA sequence encoding Clostridium toxin. It uses standard molecular cloning techniques, for example, using a short strand (oligonucleotide) of DNA encoding the desired amino acid (s) and replacing the original coding sequence with a polymerase enzyme. It can be achieved by site-directed mutagenesis or by inserting / deleting a portion of the gene using various enzymes (eg, ligase and restriction endonuclease). Alternatively, the modified gene sequence can be chemically synthesized.

In one embodiment, at least one amino acid modification is a substitution of an acidic amino acid residue with a basic amino acid residue; a substitution of an acidic amino acid residue with an uncharged amino acid residue; a substitution of an uncharged amino acid residue with a basic amino acid residue. Substitution; Insertion of Basic Amino Acid Residues; Deletion of Acidic Amino Acid Residues.

In a preferred embodiment, at least one amino acid modification is, advantageously, a substitution that maintains the same number of amino acid residues in Clostridium toxin. In one embodiment, the substitution is selected from substitution of acidic amino acid residues with basic amino acid residues, substitution of acidic amino acid residues with uncharged amino acid residues, and substitution of uncharged amino acid residues with basic amino acid residues. NS. 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 where the substitution is a substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (ie, aspartic acid is replaced by aspartic acid). Substituted, glutamic acid is replaced with glutamine).

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

The modified Clostridium toxin of the present invention comprises two or more amino acid modifications. Thus, in one embodiment, the modified Clostridium toxin (as described above) is an amino acid modification between 1 and 90 (eg, between 1 and 80, between 1 and 70, between 1 and 60, between 1 and 50). Between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10, between 3 and 50, between 3 and 40, between 3 and 30, between 4 and 40 , Amino acid modifications between 4 and 30, between 5 and 40, between 5 and 30 or between 10 and 25). In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 1 and 20. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 1 and 10. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 2 and 20. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 2 and 15. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 2 and 10. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 3 and 20. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 3 and 15. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 3 and 10. In one embodiment, the modified Clostridium toxin (as described above) comprises an amino acid modification between 4 and 40. In one embodiment, the modified Clostridium 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 modified Clostridium toxin comprises at least 3 amino acid modifications (eg, at least 3 amino acid substitutions). In one embodiment, the modified Clostridium toxin comprises at least 4 amino acid modifications (eg, at least 4 amino acid substitutions). Each of the above amino acid modifications is the above amino acid modification. Thus, each of the above amino acid modifications contributes to an increase in the pI of the modified Clostridium toxin (which lacks the amino acid modification and is otherwise compared to the pI of the same Clostridium toxin).

Any Clostridial toxin amino acid (i.e., amino acid residues) not located in a Clostridial toxin binding domain (H _C domain), as long as the modification of the result is an increase of clostridial toxin pI (as described above), as described above May be modified to. However, we have identified a subset of Clostridium toxin amino acids that are particularly suitable targets for modification.

Preferred target amino acids may have specific qualities. As an example, preferred target amino acids are (i) surface-exposed amino acids; (ii) located outside the crotridium toxin protein secondary structure; (iii) in the crotridium toxin protein region, which is not essential for protein function. Located; (iv) amino acids whose identity is not conserved between types, subtypes or serum types of crostridium toxin; (iv) amino acids whose modifications do not produce the expected ubiquitination site; or (v) It can be any combination of the above.

As mentioned above, the modified Clostridium toxins of the present invention are characterized by one or more amino acid modifications located in either the Clostridium toxin _{NN translocation domain or the Clostridium toxin light chain.}

Examples of Clostridium toxin light chain reference sequences include:
Botulinum type A neurotoxin: Amino acid residues 1-448
Botulinum type B neurotoxin: Amino acid residues 1-440
Botulinum C _{Type 1} Neurotoxin: Amino Acid Residues 1-441
Botulinum type D neurotoxin: Amino acid residues 1-445
Botulinum type E neurotoxin: Amino acid residues 1-422
Botulinum type F neurotoxin: Amino acid residues 1-439
Botulinum type G neurotoxin: Amino acid residues 1-441
Tetanus Neurotoxin: Amino Acid Residues 1-457

Examples of Clostridium toxin H _N domain reference sequences include:
Botulinum type A neurotoxin: Amino acid residues 449-871
Botulinum type B neurotoxin: Amino acid residues 443-862
Botulinum C _{Type 1} Neurotoxin: Amino Acid Residues 450-866
Botulinum type D neurotoxin: Amino acid residues 449-871
Botulinum type E neurotoxin: Amino acid residues 455-845
Botulinum type F neurotoxin: Amino acid residues 450-864
Botulinum type G neurotoxin: Amino acid residues 449-871
Tetanus Neurotoxin: Amino Acid Residues 456-879

Examples of Clostridial toxin H _C domain reference sequence, include the following:
Botulinum type A neurotoxin: Amino acid residues 872-1278
Botulinum type B neurotoxin: Amino acid residues 863 to 1291
Botulinum C _{Type 1} Neurotoxin: Amino Acid Residues 867 to 1291
Botulinum type D neurotoxin: Amino acid residues 872-1276
Botulinum type E neurotoxin: Amino acid residues 846 to 1252
Botulinum type F neurotoxin: Amino acid residues 865 to 1278
Botulinum type G neurotoxin: Amino acid residues 872-1297
Tetanus Neurotoxin: Amino Acid Residues 880-1315

The reference sequences identified above should be considered as guides, as slight variations can occur depending on the serotype subtype. As an example, US2007 / 0166332, the full text of which is incorporated herein by reference, cites a slightly different Clostridium sequence:

Light chain:
Botulinum type A neurotoxin: Amino acid residues M1-K448
Botulinum type B neurotoxin: Amino acid residues M1-K441
Botulinum C _{Type 1} Neurotoxin: Amino Acid Residues M1-K449
Botulinum type D neurotoxin: Amino acid residues M1-R445
Botulinum type E neurotoxin: Amino acid residues M1-R422
Botulinum type F neurotoxin: Amino acid residues M1-K439
Botulinum type G neurotoxin: Amino acid residues M1-K446
Tetanus neurotoxin: Amino acid residues M1-A457

H _N domain:
Botulinum type A neurotoxin: Amino acid residues A449-K871
Botulinum type B neurotoxin: Amino acid residues A442-S858
Botulinum C _{Type 1} Neurotoxin: Amino Acid Residues T450-N866
Botulinum type D neurotoxin: Amino acid residues D446-N862
Botulinum type E neurotoxin: Amino acid residues K423-K845
Botulinum type F neurotoxin: Amino acid residues A440-K864
Botulinum type G neurotoxin: Amino acid residues S447-S863
Tetanus Neurotoxin: Amino Acid Residues S458-V879

H _C domain:
Botulinum type A neurotoxin: Amino acid residues N872-L1296
Botulinum type B neurotoxin: Amino acid residues E859-E1291
Botulinum _{type 1} neurotoxin: Amino acid residues N867 to E1291
Botulinum type D neurotoxin: Amino acid residues S863-E1276
Botulinum type E neurotoxin: Amino acid residues R846-K1252
Botulinum type F neurotoxin: Amino acid residues K865-E1274
Botulinum type G neurotoxin: Amino acid residues N864 to E1297
Tetanus Neurotoxin: Amino Acid Residues I880-D1315

In one embodiment where the at least one amino acid modification is located in the Clostridium toxin translocation domain (H _N domain), the at least one amino acid modification is not located in the Clostridium toxin belt region. The Clostridium toxin belt region (as determined by visual inspection of the structure and model) is defined as:
Botulinum type A neurotoxin: Amino acid residues 492-545
Botulinum type B neurotoxin: Amino acid residues 472-532
Botulinum C _{Type 1} Neurotoxin: Amino Acid Residues 494-543
Botulinum type D neurotoxin: Amino acid residues 489-539
Botulinum type E neurotoxin: Amino acid residues 466-515
Botulinum type F neurotoxin: Amino acid residues 485-536
Botulinum type G neurotoxin: Amino acid residues 489-538
Tetanus Neurotoxin: Amino Acid Residues 506-556

In one embodiment, at least one amino acid modification (as described above) is a modification of a surface exposed amino acid residue. Surface-exposed amino acid residues are located outside the folded protein, as opposed to amino acid residues that are located inside the folded protein, and are therefore accessible to the surrounding solvent. The surface exposure of amino acid residues and thus their exposure to the surrounding solvent will vary depending on their location within the folded protein and also on the conformation taken by the protein. Therefore, modification of amino acid residues with high surface exposure can have a greater effect on the isoelectric point of a protein than modification of amino acid residues with low surface exposure. Methods for examining the surface exposure of amino acid residues are known in the art. As an example, the computer program AreaIMol (part of the CCP4 suite of computer programs) can be used to calculate the 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 (such as those provided by X-ray crystallography). In one embodiment, the surface-exposed amino acid residue has a total AreaIMol value of at least 40.

In one embodiment, at least one amino acid modification is aspartic acid residue, glutamic acid residue, histidine residue, asparagine residue, glutamine residue, serine residue, threonine residue, alanine residue, glycine residue, valine. Includes modifications of amino acid residues selected from residues, leucine residues and isoleucine residues. We have identified that amino acid residues from this group correspond to targets that are particularly suitable for the modifications of the invention.

Amino acid modifications include aspartic acid residue, glutamic acid residue, histidine residue, asparagine residue, glutamine residue, serine residue, threonine residue, alanine residue, glycine residue, valine residue, leucine residue and isoleucine. In one embodiment comprising modification of an amino acid residue selected from residues (as described above), the amino acid residue is replaced with a lysine residue or an arginine residue. In one embodiment, amino acid residues are replaced with arginine residues. Thus, in one embodiment, negatively charged residues, polar or uncharged residues are replaced with positively charged residues, thus increasing the ratio of positive to negative charges of the clostridium toxin. And increase pI.

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

In one embodiment, the modified Clostridium toxin is BoNT / A. The reference BoNT / A sequence has UniProtKB accession number P10845. In one embodiment where the modified Clostridium toxin is BoNT / A, the modified Clostridium toxin is BoNT / A1.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / A Clostridium toxin H _{N domain.}

In one embodiment where the modified Clostridium toxin is BoNT / A, the modified BoNT / A is 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, At least one selected from N752, Q753, T755, E756, E757, E758, N760, N761, I762, N763, D825, I831, G832, T847, D848 and D858 (eg, at least 1, 2, 3, 4, Includes amino acid modifications of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55; the above amino acid modifications (s) are isoelectric points (s) of the modified BoNT / A. pI) is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8) than the pI of the same BoNT / A, which lacks the amino acid modifications (s) mentioned above. , 0.9, or 1) pI units increase to higher values. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified Clostridium toxin is BoNT / A, the modified BoNT / A is N476, S564, N578, E599, L647, D650, D651, V675, I685, N687, T755, E757, N761, N763, Includes modifications of at least one (eg, at least 1, 2, 3, 4, 5, 10, 15 or 17) amino acids selected from I831, T847 and I849; said amino acid modifications (s) The isoelectric point (pI) of the modified BoNT / A is at least 0.2 (eg, at least 0.2, 0.3,) more than the pI of the same BoNT / A in other respects that lacks the amino acid modification (s). 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) Increases to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified Clostridium toxin is BoNT / A, the modified BoNT / A is at least one selected from S564, L647, D650, D651, T847 and I849 (eg, at least 1, 2, 3, Includes 4, 5 or all 6) amino acid modifications; said amino acid modifications (s) lack the isoelectric point (pI) of the modified BoNT / A and said amino acid modifications (s). In other respects, it increases by at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of the same BoNT / A. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified Clostridium toxin is BoNT / A, the modified BoNT / A is at least one selected from N476, N763, N687, E599, I831 and N761 (eg, at least 1, 2, 3, Includes 4, 5 or all 6) amino acid modifications; said amino acid modification (s) lacks the isoelectric point (pI) of the modified BoNT / A and said amino acid modification (s). In other respects, it increases by at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of the same BoNT / A. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified Clostridium toxin is BoNT / A, the modified BoNT / A is at least one selected from N578, V675, I685, T755 and E757 (eg, at least 1, 2, 3, 4 or). Includes all five) amino acid modifications; the amino acid modification (s) is the isoelectric point (pI) of the modified BoNT / A, otherwise lacking the amino acid modifications (s). Increases by at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of the same BoNT / A. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

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

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / B Clostridium toxin H _{N domain.}

In one embodiment where the modified crotridium toxin is BoNT / B, the modified BoNT / B is 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 Includes at least one amino acid modification selected from S858 (eg, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or all 40); said amino acid modification (singular) Or more) makes the isoelectric point (pI) of the modified BoNT / B at least 0.2 (eg,) more than the pI of the same BoNT / B otherwise lacking the amino acid modification (s). Increase to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment, the modified Clostridium toxin is BoNT / C ₁ . The reference BoNT / C ₁ sequence has UniProtKB accession number P18640.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / C ₁ Clostridium toxin H _{N domain.}

In one embodiment where the modified Clostridium toxin is BoNT / C ₁ , the modified BoNT / C ₁ described above is 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, At least one selected from Q681, E682, S750, G751, S759, Q760, V826, G827, N842, T843, N847 and N853 (eg at least 1, 2, 3, 4, 5, 10, 15, 20, 20) Includes amino acid modifications of 25, 30, 35, 40, 45 or 50; said amino acid modifications (s) to the isoelectric point (pI) of _{modified BoNT / C 1 as described above.} At least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI than the pI _{of the same BoNT / C 1} otherwise lacking (singular or plural) Increases to a higher value. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

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

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / D Clostridium toxin H _{N domain.}

In one embodiment where the modified crotridium toxin is BoNT / D, the modified BoNT / D is 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, E661, At least selected from T663, A666, V669, S674, I676, Q677, E678, S746, G747, D749, E751, N752, I753, Q756, N818, V822, G823, E837, N838, T839, N843, N849 and N850 Includes modification of one amino acid (eg, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 62 all); Amino acid modifications (s) make the isoelectric point (pI) of the modified BoNT / D at least more than the pIs of the same BoNT / B in other respects that lack the amino acid modifications (s). Increased to 0.2 (eg, 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment, the modified Clostridium toxin is BoNT / E. The reference BoNT / E sequence has UniProtKB accession number Q00496. In one embodiment where the modified Clostridium toxin is BoNT / E, the modified Clostridium toxin is BoNT / E3.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / E Clostridium toxin H _{N domain.}

In one embodiment where the modified Clostridium toxin is BoNT / E, the modified BoNT / E is 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, At least one selected from N645, L647, Y648, D651, E737, E741, Y803, Y824, D825, G828, I831, G832 and D835 (eg, at least 1, 2, 3, 4, 5, 10, 15, Includes amino acid modifications of 20, 25, 30, 35, 40, 45, 50 or 52; said amino acid modifications (s), said, to the isoelectric point (pI) of the modified BoNT / E. At least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) than the pI of the same BoNT / E otherwise lacking amino acid modifications (s) Increases to a higher value in pI units. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

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

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / F Clostridium toxin H _{N domain.}

In one embodiment where the modified crotridium toxin is BoNT / F, the modified BoNT / F is 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, N758, I759, N760, N761, S799, S821, I822, N840, S841, E845, L846, S847, S848, T850, N851, D852, I854, L855 and I856. One (for example, 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) Includes amino acid modifications in; said amino acid modifications (s) are identical in the isoelectric point (pI) of the modified BoNT / F, otherwise lacking the amino acid modifications (s). Increased to a value at least 0.2 (eg, 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 BoNT / F pI. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

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

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / G Clostridium toxin H _{N domain.}

In one embodiment where the modified crotridium toxin is BoNT / G, the modified BoNT / G is N480, Q482, N483, N484, T485, E487, D540, N562, N570, N571, N572, T588, V589, T615, At least one selected from D621, N637, E638, E642, N643, I660, E662, I667, E674, S675, V677, G678, N679, S747, N755, D757, L823, D839, I841, D844, S846 and L847. Includes amino acid modifications (eg, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or all 36); said amino acid modifications (s) are modified BoNT / The isoelectric point (pI) of G is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5) than the pI of the same BoNT / G otherwise lacking the amino acid modification (s). , 0.6, 0.7, 0.8, 0.9, or 1) Increase to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

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

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the TeNT Clostridium toxin H _{N domain.}

In one embodiment where the modified crotridium toxin is TeNT, the modified TeNT is 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, At least one selected from Q696, A772, D773, E774, S862, N866, L867 and D868 (eg at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, The amino acid modification (s), including all 45 or 49 amino acid modifications, lacks the isoelectric point (pI) of the modified TeNT, and otherwise lacks the amino acid modification (s). Increases by at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of the same TeNT. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / A Clostridium toxin light chain.

In one embodiment where the modified Clostridium toxin is BoNT / A, the modified BoNT / A is N5, Q7, N9, D12, N15, Q31, D58, N60, D74, N82, T122, D124, E126, Q139, At least one selected from D141, E281, L284, S295, Q311, D326, D334, N377, TYR387, N394, N396, N410, M411 and N418 (eg, at least 1, 2, 3, 4, 5, 10, Includes amino acid modifications of 15, 20, 25 or 28; said amino acid modifications (s) to the isoelectric point (pI) of the modified BoNT / A, said amino acid modifications (s). In the absence, it increases by at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of the same BoNT / A. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / B Clostridium toxin light chain.

In one embodiment where the modified crotridium toxin is BoNT / B, the modified BoNT / B is 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, At least one selected from E423 and E427 (eg, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40 or 41, including modifications of all amino acids; said amino acids The modification (s) makes the isoelectric point (pI) of the modified BoNT / B at least 0.2 than the pI of the same BoNT / B otherwise lacking the amino acid modification (s). (For example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units increase to higher values. In one embodiment, the modification is an amino acid at a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid at a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / C _{1 Clostridium toxin light chain.}

In one embodiment where the modified Clostridium toxin is BoNT / C ₁ , the modified BoNT / C ₁ described above is 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, Contains at least one amino acid selected from E427, D442 and N448 (eg, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 41); said amino acid modification (singular) Or more) make the modified BoNT / C ₁ isoelectric point (pI) at least 0.2 (or at least 0.2 (s) more than the _{same BoNT / C 1} pI in other respects lacking the amino acid modification (s). For example, increase to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / D Clostridium toxin light chain.

In one embodiment where the modified crotridium toxin is BoNT / D, the modified BoNT / D is D7, N9, D12, N15, D16, N17, D53, D73, D119, E124, E139, E142, N143, Q177, At least one selected from Q178, N180, E184, E255, N308, D335, N336, N339, N343, N368, N386, D389, D390, N391, D397, N403, N407, E409, E416 and N443 (eg at least Includes amino acid modifications of 1, 2, 3, 4, 5, 10, 15, 20, 30 or 34; the above amino acid modifications (s) are the isoelectric points (pI) of the modified BoNT / D. At least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9) than the pI of the same BoNT / D otherwise lacking the amino acid modifications (s) Or, 1) It increases to a higher value by pI unit. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / E Clostridium toxin light chain.

In one embodiment where the modified crotridium toxin is BoNT / E, the modified BoNT / E is N5, N8, N10, D11, N14, D15, Q27, E28, Q53, N72, Q75, D117, N118, D121, At least selected from N122, Q123, N138, N169, N170, N195, Q237, ILE244, Q290, N293, N297, D312, Q344, N362, N365, D366, N370, E373, N378, N379, N383, N390 and T397. Includes one (eg, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35 or all 37) amino acid modifications; the above amino acid modifications (s) are modified BoNT / The isoelectric point (pI) of E is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5) than the pI of the same BoNT / E otherwise lacking the amino acid modification (s). , 0.6, 0.7, 0.8, 0.9, or 1) Increase to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified crotridium toxin is BoNT / E, the modified BoNT / E is N5, N8, N10, D11, N14, D15, Q27, E28, N72, Q75, N118, D121, N122, Q123, Contains modification of at least one (eg, at least 1, 2, 3, 4, 5, 10, 15 or 20) amino acids selected from N138, Q237, Q290, N297, N362, N365, D366, N378 and N379. The above amino acid modification (s) is the isoelectric point (pI) of the modified BoNT / E, and the pI of the same BoNT / E is otherwise lacking the amino acid modification (s). Also increases by at least 0.2 (eg, 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified crotridium toxin is BoNT / E, the modified BoNT / E is N5, N8, N10, D11, N14, D15, N72, Q75, N118, N122, Q123, N138, Q237, Q290, Includes at least one amino acid modification selected from Q297, N362, D366, N378 and N379 (eg, at least 1, 2, 3, 4, 5, 10, 15 or 19); said amino acid modification (singular) Or more) makes the isoelectric point (pI) of the modified BoNT / E at least 0.2 (eg,) more than the pI of the same BoNT / E in other respects lacking the amino acid modification (s). Increase to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified Clostridium toxin is BoNT / E, the modified BoNT / E is at least one selected from N8, N10, Q75, Q123, N138 and Q237 (eg, at least 1, 2, 3, Includes amino acid modifications (all 4, 5 or 6); said amino acid modifications (s) lack the isoelectric point (pI) of the modified BoNT / E and said amino acid modifications (s). In other respects, it increases by at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than the pI of the same BoNT / E. In one embodiment, the modification comprises an amino acid substitution at a lysine residue or an arginine residue. In one embodiment, the modification comprises the substitution of an amino acid at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

In one embodiment where the modified crotridium toxin is BoNT / E, the modified BoNT / E modifies at least one (eg, at least 1, 2 or all 3) amino acids selected from Q123, N138 and Q237. Includes; said amino acid modification (s) is the isoelectric point (pI) of the modified BoNT / E, and lacks the said amino acid modification (s) and is otherwise identical pI of BoNT / E. Increases to a value that is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI units higher than. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / F Clostridium toxin light chain.

In one embodiment where the modified Clostridium toxin is BoNT / F, the modified BoNT / F is N6, N9, N11, D12, N15, D16, D17, E28, D55, D60, D74, N76, E105, E121, At least one selected from N126, E127, N144, D185, N211, Q252, N305, E310, D312, N314, N329, D331, N379, D382, D383, D384, E390, N396, N400, D414 and D418 (eg) , At least 1, 2, 3, 4, 5, 10, 15, 20, 30 or 35 all) amino acid modifications; said amino acid modifications (s) are isoelectric points (pI) of the modified BoNT / F. ) Is at least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, than the pI of the same BoNT / F otherwise lacking the amino acid modification (s). Increased to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the BoNT / G Clostridium toxin light chain.

In one embodiment where the modified crotridium toxin is BoNT / G, the modified BoNT / G is N4, N7, N9, N11, D12, N15, D17, E48, Q55, D57, N60, D75, D127, Q144, Selected from 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 Includes at least one amino acid modification (eg, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35 or 38); said amino acid modification (s) The Isoelectric Point (pI) of BoNT / G is at least 0.2 (eg, at least 0.2, 0.3, 0.4) than the pI of the same BoNT / G in other respects that lacks the amino acid modification (s). , 0.5, 0.6, 0.7, 0.8, 0.9, or 1) Increase to 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

We have identified specific amino acids that correspond to preferred targets for amino acid modification in the TeNT light chain.

In one embodiment where the modified crotridium toxin is TeNT, the modified TeNT is N6, N7, N15, N16, D17, D31, E51, E57, N60, N76, N101, D126, D143, N167, D179, N180, At least one selected from E251, Q257, N313, N316, D318, D335, N337, Q339, N368, N387, D390, D391, N395, D396, E403, D406, E410, N421, D427 and E450 (eg, at least Includes amino acid modifications of 1, 2, 3, 4, 5, 10, 15, 20, 30, 35 or 36; said amino acid modifications (s) have the isoelectric point (pI) of the modified TeNT. , At least 0.2 (eg, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) than the pI of the same TeNT in other respects, lacking the amino acid modifications (s) ) Increases to a higher value by pI unit. 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 at a lysine residue. In one embodiment, the modification comprises the substitution of an amino acid at an arginine residue.

The present invention is suitable for application to a large number of various Clostridium toxins. Thus, in the context of the present invention, the term "Clostridium toxin" is a toxin produced by C. botulinum (botulinum neurotoxin serum types A, B, C ₁ , D, E, F and G), C. Tetani (Genotoxin), C. butyricum (Botulinum neurotoxin serum type E) and C. baratii (Botulinum neurotoxin serum type F) and modified Clostridium toxin or any of the above. Includes derivatives derived from C. botella. The term "Clostridium toxin" also includes botulinum neurotoxin serotype H.

Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex consisting of BoNT itself complexed with several accessory proteins. Currently, there are eight different classes of botulinum neurotoxins, namely botulinum neurotoxin serotypes A, B, C ₁ , D, E, F, G and H, all of which have similar structures and effects. Share the style. Different BoNT serotypes can be distinguished on the basis of inactivation by specific neutralizing antisera, and such classification by serotype correlates with the sequence identity percentage at the amino acid level. BoNT proteins of a given serotype are further subdivided into different subtypes based on the amino acid sequence identity percentage.

After being absorbed in the gastrointestinal tract and entering the systemic circulation, BoNT binds to the presynaptic membrane of cholinergic nerve endings and blocks the release of its neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP); BoNT / C ₁ , BoNT / A and BoNT / E are synaptosome-related at 25 kDa. Cleaves protein (SNAP-25); BoNT / C ₁ cleaves syntaxin.

Clostridium tetani toxin is produced by C. tetani in a single serotype. C. butyricum produces BoNT / E, whereas C. baratii produces BoNT / F.

The term "Clostridium toxin" is also intended to include modified Clostridium toxin and its derivatives, including but not limited to those described below. The modified Clostridium toxin or derivative may contain one or more amino acids that are modified compared to the natural (unmodified) form of Clostridium toxin, or is not present in the natural (unmodified) form of Clostridium toxin. It may contain one or more inserted amino acids. As an example, a modified Clostridium toxin can have a modified amino acid sequence in one or more domains associated with a natural (unmodified) Clostridium toxin sequence. Such modifications can modify the functional aspects of the toxin, such as biological activity or persistence. Therefore, in one embodiment, the modified Clostridium toxin of the present invention is a modified modified Clostridium toxin or a modified Clostridium toxin derivative or a modified Clostridium toxin derivative.

The modified Clostridium toxin can _{have one or more modifications in the amino acid sequence of the heavy chain (such as the modified HC} domain), wherein the modified heavy chain is the target neuron. It binds with higher or lower affinity than natural (unmodified) Clostridium toxin. Such modifications in the H _C domain, in ganglioside binding site of H _C domain, or the target nerve cell ganglioside receptors and / or proteins (SV2 or synaptotagmin) that change the binding of a protein receptor in the binding site It may include modifying the residue. Examples of such modified Clostridium toxins are described in WO2006 / 027207 and WO2006 / 114308, the full text of which is incorporated herein by reference.

The modified Clostridium toxin can have one or more modifications in the amino acid sequence of the light chain, eg, modifications in the substrate binding domain or catalytic domain that can alter or modify the SNARE protein specificity of the modified LC. .. Examples of such modified Clostridium toxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference in their entirety.

The modified crostridium toxin may include one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified crostridium toxin. For example, a modified Clostridium toxin may comprise a leucine-or tyrosine-based motif, wherein the motif increases or decreases the biological activity and / or biological persistence of the modified Clostridium toxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL and xExxxLM (where x is any amino acid in the formula). Suitable tyrosine-based motifs include Y-x-x-Hy (where Hy is a hydrophobic amino acid in the formula). Examples of modified Clostridium toxins containing leucine- and tyrosine-based motifs are described in WO 2002/08268, the full text of which is incorporated herein by reference.

The term "Clostridium toxin" is intended to include hybrid and chimeric Clostridium toxins. Hybrid Clostridium toxins include at least a portion of a light chain derived from one type of Clostridium toxin or a subtype thereof and at least a portion of a heavy chain derived from another Clostridium toxin or a subtype of Clostridium toxin. In one embodiment, the hybrid Clostridium toxin may contain an entire light chain from one Clostridium toxin subtype and a heavy chain from another Clostridium toxin subtype. In another embodiment, the chimeric Clostridium toxin may contain part of a heavy chain of one type of Clostridium toxin subtype (eg, binding domain), another part of the heavy chain to another Clostridium toxin subtype. Derived from. Similarly or / or therapeutic elements may include light chain moieties derived from different Clostridium toxins. Such hybrid or chimeric crostridium toxins, for example, give the therapeutic benefit of such crostridium toxins to patients who are immunologically resistant to a given crostridium toxin subtype. Delivered to patients who may have lower than average concentrations of receptors for the chain binding domain, or to patients who may have protease resistant variants of membrane or vesicular toxin substrates (eg, SNAP-25, VAMP and syntaxin). It is useful as a means of doing so. Hybrid and chimeric Clostridium toxins are described in US 8,071,110, the full text of which is incorporated herein by reference. Thus, in one embodiment, the modified Clostridium toxin of the present invention is a modified hybrid Clostridium toxin or a modified chimeric Clostridium toxin.

The term "Clostridium toxin" is intended to include retargeted Clostridium toxin. In retargeted Clostridium toxin, Clostridium toxin is modified to contain an exogenous ligand known as the targeting moiety (TM). TM is selected to provide a binding specificity for a desired target cell as part of the retargeting process, natural binding moiety (e.g., H _C domain, or H _CC domain) of a clostridial toxin can be removed. Retargeting techniques include, for example, EP-B-0689459; WO1994 / 021300; EP-B-0939818; US6,461,617; US7,192,596; WO1998 / 007864; EP-B-0826051; US5,989,545; US6,395,513; US6,962,703; WO1996 / 033273; EP-B-0996468; US7,052,702; WO1999 / 017806; EP-B-1107794; US6,632,440; WO2000 / 010598; WO2001 / 21213; WO2006 / 059093; WO2000 / 62814; WO2000 / 04926; WO1993 / 15766; WO2000 / 61192; and WO1999 / 58571, all of which are incorporated herein by reference in their entirety. Thus, in one embodiment, the modified Clostridium toxin of the present invention is a modified retargeted Clostridium toxin.

The present invention also includes Clostridium toxins having unnatural protease cleavage sites. In such Clostridium toxins, the native protease cleavage site (also known as the activation site, as described above) is modified or modified with a protease cleavage site that is not natural for the Clostridium toxin (ie, the exogenous cleavage site). Will be replaced. Such sites require exogenous proteases for cleavage, which allows for improved control of the timing and overall location of cleavage events. Unnatural protease cleavage sites that can be used in Clostridium toxin include:
Enterokinase (DDDDK ↓)
Factor X (IEGR ↓ / IDGR ↓)
TEV (Tobacco Etch Virus) (ENLYFQ ↓ G)
Thrombin (LVPR ↓ GS)
PreScission (LEVLFQ ↓ GP).

Further protease cleavage sites include recognition sequences that are cleaved by non-cytotoxic proteases, for example, by the light chain of Clostridium neurotoxin. These include SNARE (eg, SNAP-25, syntaxin, VAMP) protein recognition sequences that are cleaved by non-cytotoxic proteases such as the light chain of Clostridium neurotoxin. Clostridium toxins containing unnatural protease cleavage sites are described in US7,132,259, EP1206554-B2 and US2007 / 0166332, all of which are incorporated herein by reference in their entirety. Intein, which is a self-cleaving sequence, is also included by the term protease cleavage site. The self-splicing reaction can be controlled, for example, by varying the concentration of the reducing agent present.

The present invention also includes Clostridium toxins containing "disruptive cleavage sites". In the Clostridium toxin, an unnatural protease cleavage site is incorporated into the Clostridium toxin at a selected location such that cleavage at the site reduces or inactivates the activity of the Clostridium toxin. The destructive protease cleavage site may be sensitive to cleavage by local proteases in the event that Clostridium toxin migrates to a non-target position after administration. Suitable non-natural protease cleavage sites include those described above. Clostridium toxins containing destructive cleavage sites are described in WO2010 / 094905 and WO2002 / 044199, both of which are incorporated herein by reference in their entirety.

The modified Clostridium toxins of the invention, in particular their light chain components, can be PEGylated (which can help increase stability, eg, the duration of action of the light chain components). Pegging is particularly preferred when the light chain contains a BoNT / A, B or C _{1 protease.} Pegging preferably involves the addition of PEG to the N-terminus of the light chain component. As an example, the N-terminus of the light chain can be extended with one or more amino acid (eg, cysteine) residues that may be the same or different. One or more of the amino acid residues may have its own PEG molecule that is attached (eg, covalently attached) to it. An example of this technique is given in WO 2007/104567, the full text of which is incorporated herein by reference.

The modified Clostridium toxins of the present invention may be free of complex-forming proteins present in naturally occurring Clostridium toxin complexes.

The modified Clostridium toxins of the invention may also contain a limited number of non-standard amino acids. Therefore, in addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine) are the amino acid residues of the modified crotridium toxin of the present invention. May be replaced with. A limited number of non-conservative amino acids, those not encoded by the genetic code and unnatural amino acids can be replaced with Clostridium polypeptide amino acid residues. The modified Clostridium toxins of the present invention may also contain non-naturally occurring amino acid residues.

Non-naturally occurring amino acids, but not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, alanine -Threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethyl homo-cysteine, nitro-glutamine, homoglutamine, pipecolinic acid, t-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine And 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, chemically aminoacylated suppressor tRNAs can be used to use in vitro systems in which nonsense mutations are suppressed. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in cell-free systems containing E. coli S30 extracts and commercially available enzymes and other reagents. The protein is purified by chromatography. For example, Robertson et al., J. Am. Chem. Soc. Vol. 113: 2722, 1991; Ellman et al., Methods Enzymol. Vol. 202: 301, 1991; Chung et al., Science Vol. 259: 806-9. See page, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA, Vol. 90: 10145-9, 1993). In the second method, translation is performed by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA in Xenopus oocytes (Turcatti et al., J. Biol. Chem. Volume 271: 19991-8, 1996). In a third method, E. coli cells are subjected to the absence of a naturally occurring amino acid to be replaced (eg, phenylalanine) and the desired non-naturally occurring amino acid (s) (eg, 2-). It is cultured in the presence of azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). Non-naturally occurring amino acids are incorporated into polypeptides in place of their naturally occurring counterparts. Koide et al., Biochem. Vol. 33: 7470-6, 1994.

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

In another aspect, the invention provides a nucleic acid (eg, DNA) comprising a nucleic acid sequence encoding a modified Clostridium toxin as described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector containing a promoter and terminator.

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

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

The nucleic acid molecule of the present invention can be made using any suitable method known in the art. Therefore, nucleic acid molecules can be made using chemical synthesis techniques. Alternatively, the nucleic acid molecule of the present invention can be made using molecular biology techniques.

The DNA constructs of the present invention are preferably designed in-computer and then synthesized by conventional DNA synthesis techniques.

The above nucleic acid sequence information is optionally modified for codon bias according to the definitive host cell (eg, E. coli) expression system to be used.

In one embodiment, the nucleic acid sequence encoding the modified crotridium toxin as described above is at least 70% (eg, at least 75, 80, 85, 90,) of the nucleic acid sequence selected from SEQ ID NOs: 2, 4 and 6. Nucleic acid sequence having 95, 97, 98, or 99%) sequence identity. In one embodiment, the nucleic acid sequence has at least 90% sequence identity to the nucleic acid sequence selected from SEQ ID NOs: 2, 4 and 6.

The present invention also provides a polypeptide encoded by a nucleic acid sequence as described above. Thus, in one aspect, the invention is at least 70% (eg, at least 75, 80, 85, 90, 95, 97, 98, or 99%) of the amino acid sequence selected from SEQ ID NOs: 1, 3 and 5. ) Provide a polypeptide containing an amino acid sequence having sequence identity. In one embodiment, the amino acid sequence has at least 90% sequence identity to the amino acid sequence selected from SEQ ID NOs: 1, 3 and 5.

In one embodiment, the modified clotridium toxin of the invention is a modified BoNT / A as described above, wherein the modified BoNT / A is at least 70 relative to the amino acid sequence selected from SEQ ID NOs: 1, 3 and 5. Includes (or consists of) an amino acid sequence having sequence identity of% (eg, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%).

In one embodiment, the modified Clostridium toxin of the invention is the modified BoNT / A as described above, wherein the modified BoNT / A comprises (or comprises) the amino acid sequence of SEQ ID NO: 1, 3 or 5.

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 a modified crotridium toxin, as described above, wherein the nucleic acid is at least 70% (eg, eg) of the nucleic acid sequence selected from SEQ ID NOs: 2, 4 and 6. Includes nucleic acid sequences with at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) sequence identity. In one embodiment, the nucleic acid comprises (or consists of) the nucleic acid sequence of SEQ ID NO: 2, 4 or 6.

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

In one embodiment, the modified Clostridium toxin of the invention is a modified BoNT / E as described above, wherein the modified BoNT / E is at least 70% (eg, at least 75, 80, 85) of SEQ ID NO: 7. , 90, 95, 97, 98, 99, 99.5, or 99.9%) contains amino acid sequences with sequence identity.

In one aspect, the invention provides a nucleic acid encoding a modified crotridium toxin as described above, wherein the nucleic acid is at least 70% relative to SEQ ID NO: 8 (eg, at least 75, 80, 85, 90, 95, Contains nucleic acid sequences having 97, 98, 99, 99.5, or 99.9%) sequence identity.

A "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by multiple sequences. Therefore,% identity can be calculated as the number of identical nucleotides / amino acids multiplied by 100 and divided by the total number of nucleotides / amino acids. The% sequence identity calculation may also take into account the number of gaps that need to be introduced to optimize the alignment of two or more sequences, and the length of each gap. Sequence comparisons and percent identity determinations between two or more sequences can be performed using specific methodological algorithms such as BLAST that will be familiar to those of skill in the art.

In one aspect, the invention provides a method of producing a single chain modified crotridium toxin protein having a light chain and a heavy chain, wherein the method comprises a nucleic acid (the nucleic acid is as described above) in a suitable host cell. It involves expressing in, lysing the host cell to provide a host cell homogenate containing the single chain modified clotridium toxin protein, and isolating the single chain modified clotridium toxin protein.

In another aspect, the invention provides a method of activating a modified crotridium toxin, which method can be obtained by the method of producing a single chain modified crotridium toxin protein as described above. And 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 making the polypeptide light and light. It involves the conversion of heavy chains into double-stranded polypeptides that are linked together by disulfide bonds.

The modified Clostridium toxins of the present invention can be used to prevent or treat certain medical or cosmetic diseases and conditions. Therefore, in a further aspect, the invention provides a modified Clostridium toxin as described above for use in pharmaceuticals.

In a related aspect, the present invention relates to strabismus, eyelid spasm, squint, dystonia (eg, spastic dystonia, stomatognathic dystonia, localized dystonia, delayed dystonia, laryngeal dystonia, limb dystonia, cervical dystonia). ), Dystonia (eg, spastic dystonia), dystonia (cosmetic) application that benefits from incapacitating cells / muscles (due to SNARE downward control or inactivation), dystonia or condition of eye movements (For example, communal squint (strabismus), vertical squint (strabismus), external straight muscle palsy, eye shake, thyroid abnormal muscle disease), spasticity, eyelid spasm, dystonia, Wilson's disease, tremor, tic, segmental myocronus, spasticity , Spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, exacerbation, back spasm, leg spasm, tension headache, levator pelvic syndrome, dichotomy, delayed dystonia, Parkinson's disease, stuttering, Unilateral facial spasticity, eyelid disorders, cerebral paralysis, localized spasms, spastic colitis, nervous bladder, anismus, limb spasticity, tics, tremor, dystonia, dystonia, acarasia, swallowing disorders, tearing, hyperhidrosis, excessive Dystonia, excessive gastrointestinal secretion, muscle pain (eg, pain from muscle spasticity), headache pain (eg, tension headache), deep wrinkles on the forehead, fine wrinkles on the skin, cancer, uterine disorders, dystonia, dystonia Provided are modified clostridial toxins as described above for use in the prevention or treatment of diseases or conditions selected from genital neurological disorders, chronic neuroinflammatory and smooth muscle disorders.

In use, the present invention uses pharmaceutical compositions comprising a modified Clostridium toxin with at least one ingredient selected from pharmaceutically acceptable carriers, excipients, adjuvants, propellants and / or salts. do.

The modified Clostridium toxins of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection can be in the form of solutions, suspensions or emulsions or dry powders that are dissolved or suspended in a suitable medium prior to use.

In the case of modified Clostridium toxin to be delivered topically, the modified Clostridium toxin may be formulated for cream (eg, for topical application) or for subcutaneous injection.

Topical delivery means include aerosols or other sprays (eg, nebulizers). In this regard, aerosol formulations of modified clotridium toxin allow delivery to the lungs and / or other nasal and / or bronchial or airway ducts.

The modified crotridium toxins of the invention can be administered to a patient by intrasubarachnoid or epidural injection in the spinal column at the level of the spinal cord segment involved in the innervation of the affected organ.

The preferred route of administration is by laparoscopic and / or localized, especially intramuscular injection.

The dosage range for administration of the modified Clostridium toxin of the present invention provides the desired therapeutic effect. The required dosage range is the exact nature of the modified Clostridium toxin or composition, the route of administration, the nature of the formulation, the age of the patient, the nature of the patient's condition, degree or severity, contraindications, if any, and the judgment of the attending physician. It will be acknowledged that it will change accordingly. These dose level variations can be adjusted using standard empirical determined methods for optimization.

A suitable daily dose (per kg of patient body weight) is 0.0001 to 1 ng / kg, preferably 0.0001 to 0.5 ng / kg, more preferably 0.002 to 0.5 ng / kg, particularly preferably 0.004 to 0.5 ng. It is in the range of / kg. Unit doses can vary from less than 1 picogram to 30 ng, but usually range from 0.01 to 1 ng per dose given less frequently, such as daily, or preferably weekly or every 6 months.

A particularly preferred dosing regimen is based on 0.05 ng of modified Clostridium toxin as a 1X dose. In this regard, preferred doses range from 1X to 100X (ie, 0.05 to 5 ng).

The fluid-administered form is usually prepared utilizing a sterile medium that is free of modified Clostridium toxins and pyrogens. The modified Clostridium toxin can be dissolved or suspended in the medium, depending on the medium and concentration used. In the preparation of the solution, the modified clotridium toxin can be dissolved in the medium, the solution is made isotonic by the addition of sodium chloride, if necessary, and sterilized by filtration through a sterile filter using sterile techniques. And then fill in a suitable sterile vial or ampoule and seal. Alternatively, if the solution stability is sufficient, the solution in the closed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or fungicides, suspensions or emulsifiers and / or topical aesthetic agents may also be dissolved in the medium.

The dry powder, which is dissolved or suspended in a suitable medium prior to use, can be prepared by filling pre-sterilized ingredients in a sterile container using sterile techniques in the sterile area. Alternatively, the components may be dissolved in a suitable container using aseptic techniques in the sterile area. The product is then lyophilized and the container is aseptically sealed.

Parenteral suspensions suitable for intramuscular, subcutaneous or intradermal injection are in substantially the same manner, except that the sterile components are suspended instead of being dissolved in a sterile medium and sterilization cannot be achieved by filtration. Prepare. Ingredients may be isolated in a sterile condition. Alternatively, after isolation, it may be sterilized by, for example, γ irradiation.

Advantageously, anti-precipitation agents, such as polyvinylpyrrolidone, are included in the composition (s) to facilitate uniform distribution of the components.

Administrations according to the invention can utilize a variety of delivery techniques, including microencapsulation, viral delivery systems or high pressure aerosol collisions.

FIG. 5 shows SDS-PAGE purification of CatH _N _v1 (Fig. 1A), CatH _N _v2 (Fig. 1B) and CatH _{N _v3 (Fig. 1C).} It is a figure which shows the SNAP-25 cutting percentage in the rat embryonic spinal nerve (eSCN) of CatH _{N _v1.} Rat embryonic spinal nerves were cultured for 3 weeks, treated with CatH _N _v1 for 24 hours, followed by Western blotting with SNAP-25-specific antibodies. The data are mean ± SEM from 3 independent experiments. It is a figure which shows the efficacy (t _{50 ) of BoNT / A and Cat H N} _v1 in a mouse phrenic nerve unilateral diaphragm assay (mPNHD). Data points are individual unilateral diaphragm preparations, mean ± SEM. CatH _N _v1 was statistically significantly slower than the reference protein BoNT / A (List Biological Laboratories). One-way ANOVA and Danette's multiple comparison test. ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 (one-way ANOVA and Danette's multiple comparison test). It is a figure which shows the SNAP-25 cutting percentage in the rat embryonic spinal nerve (eSCN) of CatH _{N _v2.} Rat embryonic spinal nerves were cultured for 3 weeks, treated with CatH _N _v2 for 24 hours, followed by Western blotting with SNAP-25-specific antibodies. The data are mean ± SEM from 3 independent experiments. It is a figure which shows the efficacy (t _{50 ) of BoNT / A and Cat H N} _v2 in a mouse phrenic nerve unilateral diaphragm assay (mPNHD). Data points are individual unilateral diaphragm preparations, mean ± SEM. CatH _N _v2 was statistically equivalent to the reference protein BoNT / A (List Biological Laboratories). One-way ANOVA and Danette's multiple comparison test. ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 (one-way ANOVA and Danette's multiple comparison test). It is a figure which shows the SNAP-25 cutting percentage in the rat embryonic spinal nerve (eSCN) of CatH _{N _v3.} Rat embryonic spinal nerves were cultured for 3 weeks, treated with CatH _N _v3 for 24 hours, followed by Western blotting with SNAP-25-specific antibodies. The data are mean ± SEM from 3 independent experiments. It is a figure which shows the efficacy (t _{50 ) of BoNT / A and Cat H N} _v3 in a mouse phrenic nerve unilateral diaphragm assay (mPNHD). Data points are individual unilateral diaphragm preparations, mean ± SEM. CatH _N _v3 was statistically significantly slower than the reference protein BoNT / A (List Biological Laboratories). One-way ANOVA and Danette's multiple comparison test. ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 (one-way ANOVA and Danette's multiple comparison test). It is a figure which shows the isoelectric focusing analysis. All three Cat H _N constructs have an observed increase in pI compared to unmodified BoNT / A. It is a figure which shows the SDS-PAGE purification of a CatLC construct. _{Catalytic activity, pEC 50} values of Cat LC compared to the BoNT / E LC reference were obtained in the BoTest A / E BoNT detection kit (BioSentinal Catalog No. A1004) according to the manufacturer's instructions. The data show mean ± standard deviation from one of three independent experiments.

arrangement

SEQ ID NO: 1. Modified BoNT / A, "CatH _N _v1", amino acid sequence.
SEQ ID NO: 2. Modified BoNT / A, "CatH _N _v1", nucleic acid sequence.
SEQ ID NO: 3. Modified BoNT / A, "CatH _N _v2", amino acid sequence.
SEQ ID NO: 4. Modified BoNT / A, "CatH _N _v2", nucleic acid sequence.
SEQ ID NO: 5. Modified BoNT / A, "CatH _N _v3", amino acid sequence.
SEQ ID NO: 6. Modified BoNT / A, "CatH _N _v3", nucleic acid sequence.
SEQ ID NO: 7. Modified BoNT / E light chain, "CatLC", amino acid sequence.
SEQ ID NO: 8. Modified BoNT / E light chain, "CatLC", nucleic acid sequence.

SEQ ID NO: 1. Modified BoNT / A, "CatH _N _v1", amino acid sequence.
MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKRRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMRYKRRFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSRDRPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 2. Modified BoNT / A, "CatH _N _v1", nucleic acid sequence.
ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAcgtCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCgtTACAAAcgtcgTTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCcgtGACcgtCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG

SEQ ID NO: 3. Modified BoNT / A, "CatH _N _v2", amino acid sequence.
MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLKKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATKAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIAKKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNKIKFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLKGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 4. Modified BoNT / A, "CatH _N _v2", nucleic acid sequence.
ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAAgAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTaAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAAgAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAAgATTAAgTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGAagGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG

SEQ ID NO: 5. Modified BoNT / A, "CatH _N _v3", amino acid sequence.
MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLKPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPKLGTFALVSYKANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYKEKEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO: 6. Modified BoNT / A, "CatH _N _v3", nucleic acid sequence.
ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTG ATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCgAtACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAAaCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT CATTCCGGAGATTGCGATCCCGaaGTTGGGTACCTTCGCGCTGGTGTCCTACAagGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACAaGGAAaAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAG TACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG

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

SEQ ID NO: 8. Modified BoNT / E light chain, "CatLC", amino acid sequence.
ATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACA TCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTCGGGATCGAGGGAAGGATTTCAGAATTCGGATCCATGCCAAAAATCAACAGCTTTAATTACAATGACCCTGTAAACGATCGTACCATCCTATACATAAAGCCGGGTGGGTGTCAAGAGTTCTACAAATCTTTCAATATTATGAAGAATATATGGATTATACCTGAGCGTAACGTTATTGGTACGACACCGCAAGATTTTCATCCACCTACTTCGTTGAAGAACGGTGACTCTTCCTATTACGACCCCAATTATCTCCAGTCGGATGAAGAGAAGGACAGATTCCTTAAAATAGTAACCAAAATCTTTAACAGGATTAATAACAATCTATCCGGAGGTATTTTGCTTGAAGAGCTTAGTAAAGCTAATCCTTACCTAGGTAACGATAATACACCAGACAACAAGTTTCATATAGGCGATGCATCCGCCGTGGAAATCAAATTTAGCAAGGGATCACAGCATATTCTCTTGCCCAACGTTATTATAATGGGGGCGGAACCAGATTTATTTGAAACAAATTCGAGTAATATTAGCCTGAGAAATAACTATATGCCGTCAAACCATGGGTTCGGTAGCATAGCGATCGTTACTTTTTCTCCCGAATACAGTTTTCGCTTCAATGATAATAGTATAAATGAGTTTATCCAAGACCCCGCACTCACGCTTATGCACGAACTCATACACTCTTTACACGGCCTGTATGGCGCTAAGGGGATAACCACTACGTGTATCATTACTCAGCAAAAGAACCCATTGATAACGAACAGGAAGGGCATTAACATCGAGGAATTTCTTACATTTGGAGGCAACGATCTGAACATTATAACTGTCGCACAGTACAATGACATCTATACCAA CTTACTAAATGATTATAGAAAAATCGCTTCTAAGTTATCCAAGGTTCAAGTCTCAAACCCTCAACTGAATCCGTATAAGGACATATTCCAAGAGAAATATGGATTAGACAAAGACGCGTCAGGAATCTATTCGGTAAACATTAACAAATTCGACGATATTTTGAAGAAACTTTACAGCTTCACGGAGTTCGACTTGGCCACCAAATTCCAGGTCAAATGCCGAGAGACATACATCGGACAGTATAAGTATTTCAAGCTGTCGAATCTCCTGAATGATTCCATATACAACATTAGTGAGGGTTACAATATAAATAACCTAAAGGTGAATTTCCGAGGCCAAAACGCCAACCTAAATCCGCGCATCATTAAACCCATCACAGGACGGGGGTTAGTGAAGAAAATAATCCGGTTTGCGGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCAC

The following examples serve to illustrate specific embodiments of the invention and never limit the scope of the invention as defined in the claims.

[Example 1]

Identification of preferred Clostridium toxin amino acids for modification.

Amino acids (mutation sites) identified as suitable candidates for modification were selected using several criteria.
1. Position of residues in the BoNT molecule (H in _N without the belt area)
2. Positions associated with secondary / tertiary structure;
3. Residue type;
4. Surface exposure.

Acidic, neutral, polar and hydrophobic residues were considered for selection.

Exposed residues were determined using AreaIMol from the CCP4 suite. Each structure was analyzed by Area IMol and the exposed residues were identified as having a total value greater than 55.

_{The secondary structure within the H N} and Te NT of each subtype was identified using the secondary structure assignment program (Stride Web Interface). Regions assigned to form α-helices, β-chains or 3 ₁₀ -helices were excluded from the selection.

Array 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.

_{Homology modeling of BoNT / C 1} , BoNT / D, BoNT / F and BoNT / G performed using LOOPP and the following sequences, respectively: P18640, P19321, YP_001390123 and Q60393.

Clostridium Toxin Preferred Clostridium Toxin Amino Acid Residues for Modification in _{H N 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, E756, 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, Q681, E682, S750, G751, S759, Q760, V826, G827, N842, T843, N847 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, E661, 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, G828, 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, 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, N758, I759, N760, N761, S799, S821, I822, N840, S841, E845, L846, S847, S848, T850, N851, D852, I854, L855 and I856

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 D868

Preferred Clostridium Toxin Amino Acid Residue for Modification in 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 modified BoNT / A molecule

Three different examples of modified BoNT / A molecules according to the present invention were produced.

A total of 55 residues were identified as candidates for mutations in the _{BoNT / AH N} domain using the method described in Example 1. The compatibility of the residues was further evaluated by visual inspection of the BoNT / A crystal structure and of 11 preferred candidates (N476, N763, N687, E599, I831, N761, N578, V675, I685, T755, E757). The list was obtained. Six additional residues were selected based on functional data showing that these residues were accepted for mutation without adversely affecting protein function. Four of these residues were in the candidate list (L647, D650, D651, T847) and two were not in the list (S564, I849). A total of 17 residues were selected for mutation, with 11 residues from the candidate list and 6 from the functional data.

Three constructs were made from the 17 residues selected: CatH _N _v1, CatH _N _v2 and CatH _N _v3. Mutations in the CatH _N construct are shown in Table 3 below:

Purification of CatH _N _v1, CatH _N _v2 and CatH _N _v3 is shown in Figures 1A, 1B and 1C, respectively.

[Example 3]

Cloning, expression and purification

A DNA construct encoding the modified BoNT / A molecule described in Example 2 was synthesized, cloned into a pJ401 expression vector and then transformed into BL21 (DE3) E. coli. This enabled soluble overexpression of recombinant modified BoNT / A molecules in E. coli.

Recombinant modified BoNT was purified from E. coli lysate using traditional chromatography techniques. The first purification step using a cation exchange resin was used, followed by an intermediate purification step using a hydrophobic interacting resin. The recombinant modified BoNT single chain was then cleaved by proteolysis, resulting in an activated double chain modified BoNT. The final purification step was then used to remove residual contaminants.

[Example 4]

Derivatization of purified modified BoNT

The modified BoNT described in Example 2 above was experimentally characterized.

The ability of modified BoNTs to invade nerves and cleave SNAP-25, the target of BoNT / A, was evaluated using rat embryonic spinal nerves (eSCNs). The efficacy of the modified BoNT was further evaluated using the mouse phrenic nerve unilateral diaphragm assay (mPNHD).

CatH _N _v1:
The first set of mutations added was substitution with arginine:
S564R, L647R, D650R, D651R, T847R, I849R

The CatH _N _v1 molecule was tested in a rat embryonic spinal cord neuron (eSCN) SNAP-25 cleavage assay and found to be as potent as BoNT / A (BoNT / A) (Fig. 2).

Positive results were also demonstrated in the mouse phrenic nerve unilateral diaphragm (mPNHD) assay (Figure 3).

CatH _N _v2:
The second set of mutations was substitution with lysine:
N476K, N763K, N687K, E599K, I831K, N761K

_{The CatH N} _v2 protein was tested in the eSCN SNAP-25 cleavage assay and found to retain the ability to invade cells and cleave SNAP-25. In the mPNHD assay, CatH _N _v2 was as potent as BoNT / A (Figures 4 and 5).

CatH _N _v3:
The third set of mutations was substitution with lysine:
N578K, V675K, I685K, T755K, E757K

_{The CatH N} _v3 molecule was tested in the eSCN SNAP-25 cleavage assay and found to retain the ability to invade cells and cleave SNAP-25. Similarly, positive results were demonstrated in the mPNHD assay (Figures 6 and 7).

Isoelectric focusing

All three Cat H _N constructs have increased pI compared to unmodified BoNT / A (Figure 8).

[Example 5]

BoNT / E light chain modification

Due to the modularity of botulinum toxin, BoNT / E light chain constructs with an N-terminal maltose-binding protein (MBP) tag and a C-terminal 6-histidine tag (6HT) are mutated and characterized. Used as a surrogate for assaying activity.

A BoNT / E light chain construct (“CatLC”) with the mutations shown in the table below was prepared.

The construct was expressed in BL21 DE3 cells and purified using affinity chromatography. Purification is shown in Figure 9.

Assessment of the construct for catalytic activity showed that the modified light chain retained the catalytic activity of the unmodified light chain (Fig. 10).

item
1. A modified crotridium toxin containing at least one amino acid modification, wherein the at least one amino acid modification lacks the isoelectric point (pI) of the modified crostridium toxin, the other point lacking the at least one amino acid modification. Increased to at least 0.2 pI units higher than the pI of the same crostridium toxin in
Wherein the at least one amino acid modification, not located in a Clostridial toxin binding domain (H _C domain), a modified Clostridial toxin.
2. The modified Clostridium toxin according to item 1, wherein at least one of the amino acid modifications is located in the Clostridium toxin translocation domain (H _{N domain).}
3. The at least one amino acid modification described above is located in the Clostridium toxin light chain,
The modified Clostridium toxin described in item 1.
4. The modified Clostridium toxin according to item 3, wherein the at least one amino acid modification described above does not introduce an E3 ligase recognition motif into the Clostridium toxin light chain.
5. The at least one amino acid modification described above makes the modified Clostridium toxin isoelectric point (pI) at least 0.5 pI than the pI of the same Clostridium toxin otherwise lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of items 1 to 4, which increases to a unit higher value.
6. At least one amino acid modification described above equals the isoelectric point (pI) of the modified Clostridium toxin, at least 1 pI unit than the pI of the same Clostridium toxin otherwise lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of items 1 to 4, which increases to a high value.
7. The at least one amino acid modification described above equals the isoelectric point (pI) of the modified Clostridium toxin, otherwise at least 2 pI units than the pI of the same Clostridium toxin lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of items 1 to 4, which increases to a high value.
8. The at least one amino acid modification described above makes the modified Clostridium toxin isoelectric point (pI) 2 to 5 more than the pI of the same Clostridium toxin otherwise lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of items 1 to 4, which increases the pI unit between high values.
9. The modified Clostridium toxin according to any of the above items, having a pI of at least 6.5.
10. The modified Clostridium toxin according to any of the above items, having a pI between 6.5 and 9.5.
11. The modified Clostridium toxin according to any of the above items, having a pI between 6.5 and 7.5.
12. The modified Clostridium toxin according to any of the above items, wherein at least one amino acid modification is selected from amino acid substitutions, amino acid insertions and amino acid deletions.
13. At least one amino acid substitution is selected from substitution of acidic amino acid residues with basic amino acid residues, substitution of acidic amino acid residues with uncharged amino acid residues, and substitution of uncharged amino acid residues with basic amino acid residues. The modified crotridium toxin according to item 12.
14. The modified Clostridium toxin according to any of the above items, which comprises an amino acid modification between 1 and 90 sites.
15. The modified Clostridium toxin according to any of the above items, which comprises at least 3 amino acid modifications.
16. The modified Clostridium toxin according to any of the above items, which comprises an amino acid modification between 4 and 40 sites.
17. The modified Clostridium toxin according to any of the above items, wherein at least one amino acid modification is a modification of a surface exposed amino acid residue.
18. At least one amino acid modification is aspartic acid residue, glutamic acid residue, histidine residue, asparagine residue, glutamine residue, serine residue, threonine residue, alanine residue, glycine residue, valine residue, The modified crotridium toxin according to any of the above items, which comprises the modification of an amino acid residue selected from a leucine residue and an isoleucine residue.
19. The modified Clostridium toxin according to item 18, wherein the amino acid residue is replaced with a lysine residue or an arginine residue.
20. A nucleic acid comprising a nucleic acid sequence encoding the modified Clostridium toxin according to any one of items 1-19.
21. A method for producing a single-chain modified clostridial toxin protein having a light chain and a heavy chain, wherein the nucleic acid according to item 20 is expressed in a suitable host cell and the host cell is lysed to simply. A method comprising providing a host cell homogenate containing a chain modified crosstridium toxin protein and isolating the single chain modified cross tridium toxin protein.
22. To provide a single-chain modified crotridium toxin protein, which is a method for activating a modified crostridium toxin, which is obtained by the method according to item 21, and a polypeptide, a polypeptide between the light chain and the heavy chain. It involves contacting a protease that cleaves at a location recognition site (cleave site) and converting the polypeptide into a double-stranded polypeptide in which the light and heavy chains are linked together by disulfide bonds. Method.
23. The modified Clostridium toxin according to any one of items 1 to 19 for use in medicine.
24. Strabismus, eyelid spasm, squint, dystonia (eg spastic dystonia, stomatognathic dystonia, localized dystonia, late-onset dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), oblique neck (eg , Spastic oblique neck), cosmetic therapy (cosmetic) application that benefits from incapacity of cells / muscles (due to SNARE downregulation or inactivation), neuromuscular disorders or conditions of eye movements (eg, strabismus) (concomitant strabismus), vertical strabismus, lateral strabismus, spasticity, thyroid abnormal muscle disease), spasticity, eyelid spasm, toothpaste, Wilson's disease, tremor, tic, segmental myocronus, spasticity, chronic multiple Spasticity due to sclerosis, spasticity resulting in abnormal bladder control, exacerbation, back spasm, leg spasm, tension headache, levator pelvic syndrome, dichotomy, delayed dyskinesia, Parkinson's disease, stuttering, unilateral facial spasm, Eyelid disorders, cerebral paralysis, localized spasticity, spasticity, spasticity, nervous bladder, anismus, limb spasticity, tics, tremor, squinting, anal cramps, acarasia, swallowing disorders, tearing, hyperperspiration, excessive saliva secretion, excess Gastrointestinal secretion, muscle pain (eg, pain from muscle spasticity), headache pain (eg, tension headache), deep wrinkles on the forehead, fine wrinkles on the skin, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, The modified strabismus toxin according to any one of items 1 to 19 for use in the prevention or treatment of a disease or condition selected from chronic neurological inflammation and spasticity disorders.
25. A modified Clostridium toxin, substantially as described herein, with reference to the accompanying drawings.
[Phase 1]
With at least one amino acid lysine or arginine selected from the group consisting of N476, S564, N578, E599, L647, D650, D651, V675, I685, N687, T755, E757, N761, N763, I831, T847, and I849. Modified botulinum toxin serotype A (BoNT / A) containing the substitution of and having an isoelectric point (pI) of at least 6.6;
here,
The at least one amino acid substitution increases the pI of the modified BoNT / A to a value at least 0.2 pI units higher than the pI of the same BoNT / A otherwise lacking the at least one amino acid substitution. ;
The modified BoNT / A retains the ability of the same BoNT / A to bind, translocate, and cleave the target SNARE protein (s) otherwise lacking the at least one amino acid substitution; And the modified BoNT / A
Encoded by a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence selected from SEQ ID NOs: 2, 4, and 6; and / or
b. Includes an amino acid sequence that has at least 90% sequence identity to the amino acid sequence selected from SEQ ID NOs: 1, 3, and 5.
[Phase 2]
The modified BoNT / A is:
Encoded by a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence selected from SEQ ID NOs: 2, 4, and 6; and / or
b. Contains an amino acid sequence that has at least 95% sequence identity to the amino acid sequence selected from SEQ ID NOs: 1, 3, and 5;
The modified BoNT / A according to embodiment 1.
[Phase 3]
The modified BoNT / A is:
Encoded by a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence selected from SEQ ID NOs: 2, 4, and 6; and / or
b. Contains an amino acid sequence that has at least 99% sequence identity to the amino acid sequence selected from SEQ ID NOs: 1, 3, and 5;
The modified BoNT / A according to embodiment 1 or 2.
[Phase 4]
The substitution increases the pI of the modified BoNT / A to a value at least 0.5 pI units higher than the pI of the same BoNT / A in other respects lacking the substitution, any of embodiments 1 to 3. Modified BoNT / A described in paragraph 1.
[Embodiment 5]
The substitution increases the pI of the modified BoNT / A to a value at least 1 pI unit higher than the pI of the same BoNT / A in other respects lacking the substitution, any one of embodiments 1-4. Modified BoNT / A described in section.
[Embodiment 6]
The substitution increases the pI of the modified BoNT / A to a value at least 2 pI units higher than the pI of the same BoNT / A in other respects lacking the substitution, any one of embodiments 1-5. Modified BoNT / A described in section.
[Phase 7]
The substitution increases the pI of the modified BoNT / A to a value in pI units between 2 and 5 that is otherwise higher than the pI of the same BoNT / A, from embodiment 1. The modified BoNT / A described in any one of 6.
[Embodiment 8]
The modified BoNT / A according to any one of embodiments 1 to 7, comprising at least 3 amino acid substitutions.
[Embodiment 9]
The modified BoNT / A according to any one of embodiments 1 to 8, comprising amino acid substitutions between 4 and 40 sites.
[Embodiment 10]
A nucleic acid comprising a nucleic acid sequence encoding the modified BoNT / A according to any one of embodiments 1-9.
[Embodiment 11]
A method for producing a single chain modified BoNT / A protein having a light chain and a heavy chain, wherein the nucleic acid according to embodiment 10 is expressed in a suitable host cell, and the host cell is lysed to obtain a simple substance. A method comprising providing a host cell homogenate containing a chain modified BoNT / A protein and isolating the single chain modified BoNT / A protein.
[Embodiment 12]
To provide a single-chain modified BoNT / A protein obtained by the method according to the eleventh embodiment, which is a method for producing a modified BoNT / A, and to obtain the single-chain modified BoNT / A protein from the single-chain modified BoNT. Contacting the / A protein with a protease that cleaves at a recognition site (cutting site) located between the light chain and the heavy chain, and combining the single chain modified BoNT / A protein with the light chain and the heavy chain by a disulfide bond. A method comprising converting to an activated double-stranded polypeptide linked to.
[Embodiment 13]
A medicament containing the modified BoNT / A according to any one of embodiments 1 to 9 or the modified BoNT / A obtained by the method according to embodiment 12.
[Phase 14]
Strabismus, palpebral spasm, squint, dystonia, spastic dystonia, spastic or oral dystonia, localized dystonia, late-onset dystonia, laryngeal dystonia, limb dystonia, cervical dystonia, squinting, spastic squinting, cells / Cosmetic therapy (cosmetic) application that benefits from incapacitating muscles (due to SNARE downward control or inactivation), neuromuscular disorders or conditions of eye movements, concomitant strabismus, vertical strabismus ), External straight muscle palsy, eye tremor, thyroid spasticity, spasticity, eyelid spasm, squinting, Wilson's disease, tremor, tic, segmental myocronus, spasticity, spasticity due to chronic multiple sclerosis, abnormal bladder control Spasticity, hate, back spasm, leg spasm, tension headache, levator pelvic syndrome, dichotomous spondylosis, late-onset dyskinesia, Parkinson's disease, stuttering, unilateral facial spasm, eyelid disorder, cerebral paralysis, localized spasticity, spasticity From colitis, nervous bladder, anismus, limb spasticity, tics, tremor, squinting, squinting, acarasia, swallowing disorders, tearing, hyperperspiration, excessive salivation, excessive gastrointestinal secretion, muscle pain, muscle spasm Prevention of diseases or conditions selected from pain, headache pain, tension headache, deep wrinkles on the forehead, fine wrinkles on the skin, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, chronic neuroinflammatory and strabismus disorders Alternatively, a medicament containing the modified BoNT / A according to any one of embodiments 1 to 9 or the modified BoNT / A obtained by the method according to embodiment 12 for use in treatment.

[Figs. 1A-C]
Marker: Marker
E. coli Lysate (soluble): E. coli lysate (solubilized)
Purification step: Purification step
[Figs. 2, 4, 6]
% SNAP-25 cleavage: SNAP-25 cleavage%
Basal: Basis
[Figs. 3, 5, 7]
t ₅₀ (min): t ₅₀ (minutes)
t ₅₀ (mean ± sem, min): t ₅₀ (mean ± SEM, minutes)
[Fig. 8]
Marker: Marker
Lane: Lane
Construct: Construct
Calculated pI: Calculated pI
Observed pI: Measured pI
[Fig. 9]
Marker: Marker
Whole cell lysate: Whole cell lysate
Capture: Capture
CatLC final: Final CatLC
[Fig. 10]
RFU ratio: RFU ratio
See Reference
Basal: Basis

Claims (25)

A modified crostridium toxin comprising at least one amino acid modification, wherein the at least one amino acid modification lacks the isoelectric point (pI) of the modified crostridium toxin, otherwise it lacks the at least one amino acid modification. Increased to at least 0.2 pI units higher than the pI of the same crostridium toxin,
Wherein the at least one amino acid modification, not located in a Clostridial toxin binding domain (H _C domain), a modified Clostridial toxin. The modified Clostridium toxin according to claim 1, wherein the at least one amino acid modification is located in the Clostridium toxin translocation domain (H _{N domain).} The at least one amino acid modification described above is located in the Clostridium toxin light chain,
The modified Clostridium toxin according to claim 1. The modified Clostridium toxin according to claim 3, wherein the at least one amino acid modification does not introduce an E3 ligase recognition motif into the Clostridium toxin light chain. The at least one amino acid modification described above raises the isoelectric point (pI) of the modified Clostridium toxin by at least 0.5 pI units higher than the pI of the same Clostridium toxin otherwise lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of claims 1 to 4, which increases in value. The at least one amino acid modification described above raises the isoelectric point (pI) of the modified Clostridium toxin by at least 1 pI unit higher than the pI of the same Clostridium toxin otherwise lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of claims 1 to 4, which is increased in number. The at least one amino acid modification described above raises the isoelectric point (pI) of the modified Clostridium toxin by at least 2 pI units higher than the pI of the same Clostridium toxin otherwise lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of claims 1 to 4, which is increased in number. The at least one amino acid modification described above equals the isoelectric point (pI) of the modified Clostridium toxin, and is otherwise between 2 and 5 more than the pI of the same Clostridium toxin lacking the at least one amino acid modification. The modified Clostridium toxin according to any one of claims 1 to 4, which increases the pI unit of the toxin to a higher value. The modified Clostridium toxin according to any of the above claims, having a pI of at least 6.5. The modified Clostridium toxin according to any of the above claims, having a pI between 6.5 and 9.5. The modified Clostridium toxin according to any of the above claims, having a pI between 6.5 and 7.5. The modified clostridial toxin according to any of the above claims, wherein at least one amino acid modification is selected from amino acid substitutions, amino acid insertions and amino acid deletions. At least one amino acid substitution is selected from substitution of acidic amino acid residues with basic amino acid residues, substitution of acidic amino acid residues with uncharged amino acid residues and substitution of uncharged amino acid residues with basic amino acid residues. , The modified crotridium toxin according to claim 12. The modified Clostridium toxin according to any of the above claims, which comprises an amino acid modification between 1 and 90 sites. The modified Clostridium toxin according to any of the above claims, which comprises at least three amino acid modifications. The modified Clostridium toxin according to any of the above claims, which comprises an amino acid modification between 4 and 40 sites. The modified Clostridium toxin according to any of the above claims, wherein at least one amino acid modification is a modification of a surface-exposed amino acid residue. At least one amino acid modification is aspartic acid residue, glutamic acid residue, histidine residue, asparagine residue, glutamine residue, serine residue, threonine residue, alanine residue, glycine residue, valine residue, leucine residue. The modified clostridial toxin according to any of the above claims, comprising modification of an amino acid residue selected from a group and an isoleucine residue. The modified Clostridium toxin according to claim 18, wherein the amino acid residue is replaced with a lysine residue or an arginine residue. A nucleic acid comprising a nucleic acid sequence encoding the modified Clostridium toxin according to any one of claims 1 to 19. A method for producing a single-chain modified crosstridium toxin protein having a light chain and a heavy chain, wherein the nucleic acid according to claim 20 is expressed in a suitable host cell, and the host cell is lysed to lyse the single chain. A method comprising providing a host cell homogenate containing a modified crotridium toxin protein and isolating a single chain modified crotridium toxin protein. To provide a single-chain modified crotridium toxin protein, which is a method for activating a modified crostridium toxin, which is obtained by the method according to claim 21, and a polypeptide is located between the light chain and the heavy chain. A method comprising contacting a protease that cleaves at a recognition site (cleave site) to cleave and converting the polypeptide into a double-stranded polypeptide in which the light and heavy chains are linked together by a disulfide bond. .. The modified Clostridium toxin according to any one of claims 1 to 19 for use in pharmaceuticals. Strabismus, eyelid spasm, squint, dystonia (eg spastic dystonia, stomatognathic dystonia, localized dystonia, late-onset dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), oblique neck (eg spasticity) Incapacity of cells / muscles (by SNARE downregulation or inactivation), cosmetic therapy (cosmetic) applications, neuromuscular disorders or conditions of eye movements (eg, concomitant) strabismus), vertical strabismus, lateral straight muscle palsy, eye tremor, thyroid abnormal muscle disease), spasticity, eyelid spasm, tooth grind, Wilson's disease, tremor, tic, segmental myocronus, spasticity, chronic multiple sclerosis Spasticity, spasticity resulting in abnormal bladder control, exacerbation, back spasm, leg spasm, tension headache, levator pelvic syndrome, dichotomy, delayed dyskinesia, Parkinson's disease, stuttering, unilateral facial spasm, eyelid disorder , Cerebral paralysis, localized spasticity, spastic colitis, nervous bladder, anismus, limb spasticity, tic, tremor, strabismus, spasticity, acarasia, swallowing disorder, tearing, hyperperspiration, excessive salivation, excessive gastrointestinal Secretory, muscle pain (eg, pain from muscle spasticity), headache pain (eg, tension headache), deep wrinkles on the forehead, fine wrinkles on the skin, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, chronic nerves The modified strabismus toxin according to any one of claims 1 to 19 for use in the prevention or treatment of a disease or condition selected from spastic inflammation and spasticity disorders. A modified Clostridium toxin, substantially as described herein, with reference to the accompanying drawings.

Images