JP2019206582A - Cationic neurotoxins - Google Patents

Abstract

To provide engineered clostridial toxins which have properties of increased tissue retention at the site of administration.SOLUTION: An engineered clostridial toxin comprises at least one amino acid modification. The at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI unit higher than the pI of an otherwise identical clostridial toxin lacking the at least one amino acid modification, where the at least one amino acid modification is not located in the clostridial toxin binding domain (Hdomain). A medical use of the engineered clostridial toxin is also provided.SELECTED DRAWING: Figure 2

Description

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

  Clostridia bacteria produce specific protein toxins that are highly potent that can be toxic to the nerves and other cells to which they are delivered. Examples of such clostridial toxins include neurotoxin produced by C. tetani (TeNT) and neurotoxin produced by C. botulinum (BoNT) serotypes A to G and C. barati ( baratii) and C. butyricum.

Among the clostridial toxins are some of the most potent toxins known. By way of example, a botulinum neurotoxin has a mouse half-lethal dose (LD ₅₀ ) value ranging from 0.5 to 5 ng / kg depending on the serotype. Both tetanus toxin and botulinum toxin act by inhibiting the function 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 tetanus toxin acts in the central nervous system.

In fact, clostridial toxins are synthesized as single chain polypeptides that are post-translationally modified by a cleavage event due to protein cleavage to form two polypeptide chains linked together by disulfide bonds. 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 the heavy chain (H chain) with a molecular mass of approximately 100 kDa and the light chain (L chain) with a molecular mass of approximately 50 kDa. The H chain includes 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 internalization of the toxin to cells bound by the binding and endosomal with its target neurons H _C domain, H _N domain is translocated into the cytosol of the L chain across the endosomal membrane, L The chain provides protease function (also known as non-cytotoxic protease).

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

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

By way of example, the inventors have described Botulinum Neurotoxin (BoNT), BoNT / A, BoNT / B, BoNT / C ₁ , to inhibit neurotransmission in several therapeutic and cosmetic or aesthetic applications. Literature describing the use of clostridial 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, commercially available botulinum toxin products are localized spasticity, upper limb spasticity, lower limb spasticity, cervical dystonia, blepharospasm, unilateral facial convulsions, excessive sweating of the eyelids, chronic migraine, neurogenic detrusor overactivity, the line between the eyebrows And currently approved for the treatment of indications involving severe unilateral eye angle lines. In addition, clostridial toxin therapy is used to treat neuromuscular disorders (see (US 6,872,397)); to treat uterine disorders (see (US 2004/0175399)); ulcers and gastroesophageal tract To treat reflux disease (see (US2004 / 0086531)); to treat dystonia (see (US6,319,505)); to treat eye disorders ((US2004 / 0234532 ); To treat blepharospasm (see (US2004 / 0151740)); to treat strabismus (see (US2004 / 0126396)); treat pain (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 injury (see (US6,423,319)); To treat rhinitis headache ((US6,838, 434); to treat tension headache (see (US6,776,992)); to treat headache (see (US6,458,365)); migraine pain (See (US5,714,469)); to treat cardiovascular disease (see (US6,767,544)); to treat neurological diseases such as Parkinson's disease (( (See US6,620,415), (US6,306,403)); to treat neuropsychiatric disorders (see (US2004 / 0180061), (US2003 / 0211121)); to treat endocrine disorders ( (See (US 6,827,931)); to treat thyroid disorders (see (US 6,740,321)); to treat sweat gland disorders affected by choline action (see (US 6,683,049)) (See (US 6,337,075), (US 6,416,765)); to treat pancreatic disorders (see (US 6,261,572), (US 6,143,306)) To treat cancers such as bone tumors ((US6,565,870 ), (US 6,368,605), (US 6,139,845), (US 2005/0031648)); to treat ear disorders (see (US 6,358,926), (US 6,265,379)) To treat autonomic disorders such as gastrointestinal myopathy and other smooth muscle dysfunctions (see (US5,437,291)); for the treatment of skin lesions associated with skin cell proliferative disorders (( (See US5,670,484)); for the management of neuroinflammatory disorders (see (US6,063,768)); for reducing hair loss and stimulating hair growth (see (US6,299,893)) For the treatment of bent-down mouths (see (US 6,358,917)); to reduce appetite (see (US 2004/40253274)); for dental and dental procedures To treat neuromuscular disorders and conditions (see (US2002 / 0010138)); to treat various disorders and conditions and associated pain (see (US2004 / 0115139)) See (US2004 / 0013692) To treat conditions caused by mucus hypersecretion such as asthma and COPD (see (WO00 / 10598)); and inflammation, endocrine, exocrine, immunological, cardiovascular It has been described (see (WO01 / 21213)) to treat non-neurological conditions such as conditions, bone conditions and the like. All of the above publications are incorporated herein by reference in their entirety.

  The use of non-cytotoxic proteases such as clostridial toxins (e.g., BoNT and TeNT) in the therapeutic and cosmetic treatment of humans and other mammals is a growing range of diseases that can benefit from the properties of these toxins And is expected to spread to the disease.

  In order to avoid systemic neurological effects, many clostridial toxin therapies involve direct administration of a clostridial toxin therapeutic to a given target site (such as a target tissue). A problem with administering clostridial toxin-based therapeutics in this manner is the spread of the toxin to the surrounding tissue or systemic circulation away from the site of administration. The diffusion of toxins away from the target tissue is thought to cause undesirable side effects that, in extreme cases, can be life-threatening. This can be a particular concern when using clostridial toxin therapeutics (such as BoNT therapeutics) at high doses, concentrations and injection volumes. Adverse effects associated with this problem reported for over-the-counter BoNT / A treatments include asthenia, generalized muscle weakness, double vision, drooping eyelids, dysphagia, dysphonia, articulation disorders, urinary incontinence and dyspnea Can be mentioned. Swallowing and dyspnea can be life threatening and deaths associated with the spread of toxic 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)

  Accordingly, there is a need in the art for a clostridial toxin that has the property of increased retention in tissue at the site of administration compared to known clostridial toxins and thus exhibits reduced diffusion away from the site of administration.

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

In one aspect, the invention provides a modified Clostridial toxin comprising at least one (e.g., at least one, two or three) amino acid modification, wherein said at least one amino acid modification is a modified Clostridial The isoelectric point (pI) of the toxin is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, less than the pI of the same clostridial toxin, which lacks at least one amino acid modification as 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, "is not located in the Clostridial toxin binding domain (H _C) domain", that at least one amino acid modification of the is located in the Clostridial toxin H _N domain or in a clostridial toxin light chain means.

In one embodiment, the invention provides a modified Clostridial toxin comprising at least one (e.g., at least 1, 2, or 3) amino acid modification, wherein said at least one amino acid modification is a modified Clostridial The isoelectric point (pI) of the toxin is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, less than the pI of the same clostridial toxin, which lacks at least one amino acid modification as described above. 0.7, 0.8, 0.9, or 1) Increased to a higher pI unit, the at least one amino acid modification is located in the clostridial toxin translocation domain (H _N domain).

  In another embodiment, the invention provides a modified Clostridial toxin comprising at least one (e.g., at least one, two, or three) amino acid modification, wherein said at least one amino acid modification is altered. The isoelectric point (pI) of the clostridial toxin is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6 0.7, 0.8, 0.9, or 1) increased to a high value of pI units, said at least one amino acid modification being located in the clostridial toxin light chain.

  In one embodiment, the at least one amino acid modification is located in a clostridial toxin light chain, and the at least one amino acid modification does not introduce an E3 ligase recognition motif in the clostridial toxin light chain. Thus, in one embodiment, the light chain of the modified Clostridial 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 neurotoxin polypeptide by the endogenous proteasome degradation pathway present in the subject to which the neurotoxin has been applied. Point to. The “E3 ligase recognition” motif is the recognition and motif of motifs by E3 ligase (also known as E3 ubiquitin ligase; therefore, “E3 ligase recognition motif” may also be referred to as “E3 ubiquitin ligase recognition motif”) It is a structural motif that enables binding. 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 include ETFSDLWKLLPE (SEQ ID NO: 20), TSFAEYWNLLSP (SEQ ID NO: 21), LTFEHYWAQLTS (SEQ ID NO: 22), LTFEHWWAQLTS (SEQ ID NO: 23), LTFEHSWAQLTS (SEQ ID NO: 24), ETFEHNWAQLTS (SEQ ID NO: 25) , LTFEHNWAQLTS (SEQ ID NO: 26), LTFEHWWASLTS (SEQ ID NO: 27), LTFEHWWSSLTS (SEQ ID NO: 28), LTFTHWWAQLTS (SEQ ID NO: 29), ETFEHWWAQLTS (SEQ ID NO: 30), LTFEHWWSQLTS (SEQ ID NO: 31), LTFEHWWAQLLS (SEQ ID NO: 32), ETFEHWWSQLLS (SEQ ID NO: 33), RFMDYWEGL (SEQ ID NO: 34), MPRFMDYWEGLN (SEQ ID NO: 35), SQETFSDLWKLLPEN (SEQ ID NO: 36) and LTFEHNWAQLEN (SEQ ID NO: 37).

  In one embodiment, wherein the at least one amino acid modification is located in a clostridial toxin light chain, the at least one amino acid modification does not introduce an MDM2 E3 ligase recognition motif into the clostridial toxin light chain. Thus, in one embodiment, the light chain of the modified Clostridial toxin of the present invention does not contain an MDM2 E3 ligase recognition motif.

  In one embodiment, wherein the at least one amino acid modification is located in a clostridial toxin light chain, the modified clostridial toxin does not include an amino acid modification in the N-terminal proline.

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

  In one embodiment, wherein the modified Clostridial toxin is BoNT / E and the at least one amino acid modification is located in the Clostridial toxin light chain, the modified BoNT / E comprises the following amino acid positions: Q53, N72, N378 , N379, or T400 does not include substitution with lysine.

  In one embodiment, wherein the modified Clostridial toxin is BoNT / E and the at least one amino acid modification is located in the Clostridial toxin light chain, the modified BoNT / E comprises the following amino acid positions: Q53, N72, N378 , N379, R394 and T400 do not include substitution with lysine.

  In one embodiment, wherein the modified Clostridial toxin is BoNT / E and the at least one amino acid modification is located in the Clostridial toxin light chain, the modified BoNT / E comprises 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 clostridial toxin light chain, the modified clostridial toxin is not BoNT / E.

A modified Clostridial toxin of the present invention does not contain any amino acid modification located in the Clostridial 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, wherein the at least one amino acid modification is located in a clostridial toxin light chain, the at least one amino acid modification does not include a substitution of an amino acid residue with a lysine residue.

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

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

  In one embodiment, wherein the modified Clostridial toxin is a modified Clostridial toxin as described above, said at least one amino acid modification lacks the pI of the modified Clostridial toxin, otherwise lacks said at least one amino acid modification. Increase to at least 0.4 pI units higher than the pI of the same clostridial toxin. In one embodiment, the at least one amino acid modification has a pI of the modified Clostridial toxin that is at least 0.5 pI units higher than the pI of the same Clostridial toxin that lacks the at least one amino acid modification, or otherwise. Increase to value. In one embodiment, the at least one amino acid modification has a pI of the modified Clostridial toxin that is at least 0.6 pI units higher than the pI of the same Clostridial toxin, otherwise lacking the at least one amino acid modification. Increase to value. In one embodiment, the at least one amino acid modification has a pI of the modified Clostridial toxin that is at least 0.8 pI units higher than the pI of the same Clostridial toxin that lacks the at least one amino acid modification and otherwise. Increase to value. In one embodiment, the at least one amino acid modification has a pI of the modified Clostridial toxin that is at least 1 pI unit higher than the pI of the same Clostridial toxin otherwise lacking the at least one amino acid modification. To increase.

  In certain embodiments, the modified Clostridial 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, said at least one amino acid modification is at least a few of the pI of the modified Clostridial toxin, but is otherwise less than the pI of the same Clostridial toxin that lacks said at least one amino acid modification. Increase to 4 or 5 pI units higher.

  In certain embodiments, the modified Clostridial toxin comprises at least three amino acid modifications, wherein said at least three amino acid modifications lack the pI of the modified Clostridial toxin, otherwise lack said at least three amino acid modifications. Increase to at least 0.2 pI units higher than the pI of the same clostridial toxin.

  In certain embodiments, the modified Clostridial toxin comprises at least 5 amino acid modifications, wherein the at least 5 amino acid modifications lack the pI of the modified Clostridial toxin, otherwise lack the at least 5 amino acid modifications. Increase to at least 0.5 pI units higher than the pI of the same clostridial toxin.

  We have increased the pI of clostridial toxins, for example by increasing at least 0.2 pI units or 0.5 pI units or 1 pI units (by introducing at least one amino acid modification into the clostridial toxin protein). The toxin advantageously demonstrates the properties of increased tissue retention and reduced diffusion away from the administration site while retaining the ability to target cell binding, translocation and cleavage of the target SNARE protein (s). I found out. Thus, the spread of clostridial toxin from the site of administration is greatly reduced compared to the otherwise identical clostridial toxin, which lacks said at least one amino acid modification.

  The modified clostridial toxins of the present invention are suitable for use in any of the above therapies and may advantageously demonstrate reduced or no side effects compared to the use of known clostridial toxin therapeutics. The increased tissue retention properties of the modified Clostridial toxins of the present invention also provide increased potency and / or duration of action compared to known Clostridial toxin therapeutics, with reduced dosage (or further adverse effects). Not increased doses) can be used, thus providing further advantages.

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

  As discussed in more detail below, the increase in pI provided by at least one amino acid modification indicates that the modified Clostridial toxin of the present invention lacks said at least one amino acid modification at a given pH, etc. Means that it has a net charge that is more positive than the net charge of the same clostridial toxin. Without wishing to be bound by any one theory, the inventors have shown that this increased positive charge makes the modified Clostridial toxin of the present invention at the site of administration modified Clostridial toxin and anionic cells. It is believed that favorable electrostatic interactions between outer components (such as cell membranes and heparin sulfate proteoglycans) allow for longer tissue retention times at the site of administration. These improved electrostatic interactions serve to reduce the diffusion of modified Clostridial toxins away from the administration site and thus improve tissue retention.

  By way of example, the improved tissue retention properties of the modified Clostridial toxins of the present invention include (i) higher doses to individual muscles such as the sternocleidomastoid muscle that do not spread to adjacent muscles in the neck causing dysphagia And (ii) 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. Benefits for patients include more effective treatment of large muscles such as the sternocleidomastoid muscle, increased chances of injection into several different muscles between each treatment, and longer duration of effective treatment with higher dosages (more Long (until re-treatment is needed).

  In one embodiment, a modified Clostridial toxin of the present invention has a positive net charge in use (eg, when the modified Clostridial toxin is located at a desired administration site in tissue 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 various side chains (or R groups), meaning that each amino acid residue in the 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 vary depending on the sum of these various factors.

  Certain amino acid residues (detailed below) have ionizable side chains that can be charged depending on the surrounding pH. Whether such a side chain is charged at a given pH depends on the pKa of the relevant ionizable moiety, where pKa is the acid dissociation constant (Ka) of a particular proton derived from the conjugate base. 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 ionizable group ). Therefore, these side chains are negatively charged at a pH of 7.4 (often referred to as “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 groups with a pKa value of approximately 10-12. These side chains are therefore positively charged at a pH of 7.4. These side chains are deprotonated at high pH values and lose their charge.

  Thus, the overall (net) charge of a protein molecule varies depending on the number of acidic and basic residues present in the protein (and its surface exposure) and on the surrounding pH. Changing the ambient pH changes the overall charge on the protein. Thus, 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 familiar to those skilled in the art.

  The isoelectric point (pI) is therefore 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 zero net charge. Thus, 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 zero net charge. Thus, a reduction in pI represents a reduction in the net positive charge of the protein at a given pH.

  Methods for determining the pI of a protein are known in the art and are well known to those skilled 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; preferred computer programs for calculating pI values include Protein Calculator from Scripps Research Institute and Compute pI / from ExPASy. MW Tool. 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 by experimentation using isoelectric focusing techniques (“actual pI”). This technique uses electrophoresis to separate proteins according to their pI. Isoelectric focusing is usually performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein is moved by the pH gradient until it reaches a pH with zero net charge, which is the pI of the protein. The results provided by isoelectric focusing are usually of relatively low resolution in nature and we have found that the results provided by the calculated pI (as described above) are more appropriate to use. I believe that.

  Throughout this specification, “pI” means “calculated pI” unless otherwise specified.

  The pI of a protein can be increased or decreased by changing the number of basic and / or acidic groups presented on its surface. This can be achieved by modifying one or more amino acids of 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) clostridial toxin has a pI of approximately 5-6. Thus, at a pH of 7.4, 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 at pH 7.4 of -8. These pI values are calculated as described above.

  As described above, in one embodiment, the modified Clostridial 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 Clostridial toxin, The absence of said at least one amino acid modification otherwise increases to a value at least 0.2 pI units higher than the pI of the same clostridial toxin.

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

  As described above, in one embodiment, the modified Clostridial 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 Clostridial toxin, The absence of said at least one amino acid modification otherwise increases to a value at least 1 pI unit higher than the pI of the same clostridial toxin.

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

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

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

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

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

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

  As discussed above, the modified Clostridial toxins of the present invention also provide increased potency and / or duration of action compared to known Clostridial toxin therapeutics, with reduced dosage (or further adverse effects). (Not increased dose) has increased tissue retention properties that can be used. One way in which these advantageous properties (corresponding to increased therapeutic indices) can be defined is the safety factor of the modified Clostridial toxin. In this context, the undesirable effects of clostridial toxins (caused by the diffusion of toxins away from the administration site) are associated with weight loss in related animal models (e.g. mice in which weight loss is detected within 7 days of administration). Can be evaluated experimentally by measuring the percentage of Conversely, the desired on-target effect of a clostridial toxin can be assessed experimentally by the Digital Abduction Score (DAS) assay, a measure of muscle paralysis. The DAS assay consists of injection of 20 μl of a clostridial toxin formulated in Gelatin Phosphate Buffer into a mouse gastrocnemius / soleus muscle complex, followed by Aoki's method (Aoki KR, Toxicon 39: 1815-1820). ; 2001) can be implemented by evaluation of the Digital Abduction Score. In the DAS assay, mice are suspended for a short period of time by their tails to elicit a characteristic startle response that stretches their hind limbs and then abducts their fingers. After clostridial toxin injection, the varying degree of finger abduction is scored on a 5-point scale (0 = normal to 4 = maximum reduction of finger abduction and limb extension).

  The clostridial toxin safety rate is then determined by 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. Thus, a high safety factor score is desired, indicating a toxin that can efficiently numb the target muscle with little undesirable off-target effects. The modified toxins of the present invention have a safety factor that is higher than that of an equivalent unmodified (natural) botulinum toxin.

Thus, in one embodiment, a modified Clostridial toxin of the invention has a safety factor of at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50), Here, the safety factor is calculated as the dose of toxin required for -10% body weight change (pg / mouse) divided by DAS ED ₅₀ (pg / mouse) [ED ₅₀ yields a DAS score of 2 Is the dose needed to].

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

  The modified Clostridial toxins of the present invention contain at least one amino acid modification. Said at least one amino acid modification increases the pI of a clostridial toxin, as discussed above. In the context of the present invention, an amino acid modification is a modification of the amino acid sequence of a clostridial toxin. Such modifications can be accomplished by replacing one amino acid in the sequence with another (ie, substitution), inserting a new amino acid in the sequence, or deleting an amino acid in the sequence. Amino acids that are incorporated into amino acid sequences in proteins are also referred to as 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 negative charges, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) are positive. Have 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 that 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.

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

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

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

  Alternatively, the substituted amino acid in an amino acid substitution can be a non-standard amino acid (amino acids that are not part of the 20 standard sets described above). By way of example, the substituted amino acid may be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid or the D 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.

  In an amino acid insertion, additional amino acid residues (those that are not normally present) are incorporated into the Clostridial toxin amino acid sequence, thus increasing the total number of amino acid residues in the sequence. Amino acid deletion removes amino acid residues from a Clostridial toxin amino acid sequence, thus reducing the total number of amino acid residues in the sequence.

  Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. As an example, amino acid modifications can be introduced by modification of a DNA sequence encoding a clostridial toxin. This is done using standard molecular cloning techniques, for example using a short strand of DNA (oligonucleotide) encoding the desired amino acid (s) and using polymerase enzyme to replace the original coding sequence. Can be accomplished by site-directed mutagenesis, or by inserting / deleting a portion of the gene using various enzymes (eg, ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized.

  In one embodiment, the 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 non-charged amino acid residue with a basic amino acid residue Selected from: substitution; insertion of a basic amino acid residue; deletion of an acidic amino acid residue.

  In preferred embodiments, the at least one amino acid modification is advantageously a substitution that maintains the same number of amino acid residues in the clostridial toxin. In one embodiment, the substitution is selected from substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, substitution of an uncharged amino acid residue with a basic amino acid residue. The 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 (i.e., aspartic acid is replaced with asparagine). 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 Clostridial toxins of the present invention contain two or more amino acid modifications. Thus, in one embodiment, a modified Clostridial toxin (as described above) has between 1 and 90 amino acid modifications (e.g. between 1 and 80, between 1 and 70, between 1 and 60, between 1 and 50). Between 1-40, between 1-30, between 1-20, between 1-10, between 3-50, between 3-40, between 3-30, between 4-40 , Between 4 and 30, between 5 and 40, between 5 and 30 or between 10 and 25 amino acid modifications). In one embodiment, the modified Clostridial toxin (as described above) comprises between 1 and 20 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 1 and 10 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 2 and 20 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 2 and 15 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 2 and 10 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 3 and 20 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 3 and 15 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 3 and 10 amino acid modifications. In one embodiment, the modified Clostridial toxin (as described above) comprises between 4 and 40 amino acid modifications. In one embodiment, the modified Clostridial toxin comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid modifications. In one embodiment, the modified Clostridial toxin comprises at least 3 amino acid modifications (eg, at least 3 amino acid substitutions). In one embodiment, the modified Clostridial toxin comprises at least 4 amino acid modifications (eg, at least 4 amino acid substitutions). Each of the amino acid modifications is an amino acid modification as described above. Thus, each of the amino acid modifications contributes to an increase in the pI of the modified Clostridial toxin (which lacks the amino acid modification, otherwise compared to the pI of the same Clostridial 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. However, the inventors have identified a subset of clostridial toxin amino acids that are particularly suitable targets for modification.

  Preferred target amino acids may have a certain quality. By way of example, preferred target amino acids are (i) amino acids exposed on the surface; (ii) located outside the clostridial toxin protein secondary structure; (iii) in a clostridial toxin protein region that is not essential for protein function (Iv) an amino acid whose identity is not conserved between Clostridial toxin types, subtypes or serotypes; (iv) an amino acid whose modification does not create the expected ubiquitination site; or (v) It can be any combination of the above.

As described above, a modified Clostridial toxin of the present invention is characterized by one or more amino acid modifications located either Clostridial toxin H _N translocation domain or a Clostridial toxin light chain.

Examples of clostridial toxin light chain reference sequences include the following:
Botulinum type A neurotoxin: amino acid residues 1-448
Botulinum type B neurotoxin: amino acid residues 1 to 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 to 439
Botulinum G-type neurotoxin: amino acid residues 1-441
Tetanus neurotoxin: amino acid residues 1 to 457

Examples of Clostridial toxin H _N domain reference sequence, include the following:
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 sequences include:
Botulinum type A neurotoxin: amino acid residues 872-1278
Botulinum type B neurotoxin: amino acid residues 83-1291
Botulinum C type ₁ neurotoxin: amino acid residues 867-1291
Botulinum type D neurotoxin: amino acid residues 872-1276
Botulinum type E neurotoxin: amino acid residues 846-1252
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

  Since slight variations can occur according to serotype subtype, the reference sequence identified above should be considered as a guide. As an example, US2007 / 0166332, which is incorporated herein by reference in its entirety, cites slightly different clostridial sequences:

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 to K439
Botulinum G-type neurotoxin: amino acid residues M1 to K446
Tetanus neurotoxin: amino acid residues M1-A457

H _N domain:
Botulinum type A neurotoxin: amino acid residues A449 to 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 to N862
Botulinum type E neurotoxin: amino acid residues K423 to K845
Botulinum type F neurotoxin: amino acid residues A440 to K864
Botulinum type G neurotoxin: amino acid residues S447 to S863
Tetanus neurotoxin: amino acid residues S458 to V879

H _C domain:
Botulinum type A neurotoxin: amino acid residues N872 to L1296
Botulinum type B neurotoxin: amino acid residues E859 to E1291
Botulinum C type ₁ neurotoxin: amino acid residues N867-E1291
Botulinum type D neurotoxin: amino acid residues S863 to E1276
Botulinum type E neurotoxin: amino acid residues R846 to K1252
Botulinum type F neurotoxin: amino acid residues K865-E1274
Botulinum type G neurotoxin: amino acid residues N864-E1297
Tetanus neurotoxin: amino acid residues I880-D1315

At least one amino acid modification of the can, in one embodiment located in the Clostridial toxin translocation domain (H _N domain), of at least one amino acid modification of the are not located in the clostridial toxin belt region. The clostridial 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, the at least one amino acid modification (as described above) is a modification of a surface exposed amino acid residue. Surface exposed amino acid residues are external to the folded protein as opposed to amino acid residues located in the interior of the folded protein, and are therefore accessible to the surrounding solvent. The surface exposure of the amino acid residue and thus its exposure to the surrounding solvent will vary depending on its position in the folded protein and also the conformation taken by the protein. Therefore, modification of amino acid residues having a high degree of surface exposure can have a greater effect on the isoelectric point of the protein than modification of amino acid residues having a low degree of surface exposure. Methods for determining 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 that provided by X-ray crystallography). In one embodiment, the surface exposed amino acid residues have a total AreaIMol value of at least 40.

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

  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 and isoleucine In one embodiment involving modification of an amino acid residue selected from residues (as described above), the amino acid residue is substituted with a lysine residue or an arginine residue. In one embodiment, amino acid residues are substituted with arginine residues. Thus, in one embodiment, a negatively charged residue, a polar residue or an uncharged residue is replaced with a positively charged residue, thus increasing the positive to negative charge ratio of the clostridial toxin. And increase pI.

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

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

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of BoNT / A clostridial toxin H _N domain.

  In one embodiment, where the modified Clostridial toxin is BoNT / A, said 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 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55) amino acid modifications; said amino acid modification (s) include the isoelectric point of the modified BoNT / A ( pI) is at least 0.2 (e.g. at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8) than the pI of the otherwise identical BoNT / A, which lacks the amino acid modification (s) described above. , 0.9, or 1) pI single To increase to a higher value. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  In one embodiment where the modified Clostridial toxin is BoNT / A, said modified BoNT / A is N476, S564, N578, E599, L647, D650, D651, V675, I685, N687, T755, E757, N761, N763, Comprising at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15 or 17 all) amino acid modification selected from I831, T847 and I849; said amino acid modification (s) The isoelectric point (pI) of the modified BoNT / A is at least 0.2 (e.g., at least 0.2, 0.3, less than the pI of the same BoNT / A, otherwise lacking the amino acid modification (s) described above. 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) Increase to a higher value in pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  In one embodiment, where the modified Clostridial toxin is BoNT / A, said modified BoNT / A is at least one selected from S564, L647, D650, D651, T847 and I849 (e.g., at least 1, 2, 3, (4, 5 or 6) amino acid modifications; said amino acid modification (s) lacks the isoelectric point (pI) of the modified BoNT / A and lacks said amino acid modification (s) In other respects, it increases to 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 a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  In one embodiment where the modified Clostridial toxin is BoNT / A, the modified BoNT / A is at least one selected from N476, N763, N687, E599, I831 and N761 (e.g., at least 1, 2, 3, (4, 5 or 6) amino acid modifications; the amino acid modification (s) lacks the isoelectric point (pI) of the modified BoNT / A and lacks the amino acid modification (s) In other respects, it increases to 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 a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  In one embodiment, where the modified Clostridial toxin is BoNT / A, said modified BoNT / A is at least one selected from N578, V675, I685, T755 and E757 (e.g., at least 1, 2, 3, 4, or All five) amino acid modifications; wherein said amino acid modification (s) are the isoelectric point (pI) of the modified BoNT / A, otherwise lacking said amino acid modification (s) Increases 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 pI of the same BoNT / A. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of BoNT / B Clostridial toxin H _N domain.

  In one embodiment where the modified Clostridial 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 Including at least one amino acid modification selected from S858 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40); Or) has an isoelectric point (pI) of the modified BoNT / B that is at least 0.2 (e.g., less than the pI of the same BoNT / B otherwise lacking the amino acid modification (s) above. 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 a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

In one embodiment, a modified Clostridial toxin is a BoNT / C _1. Referring BoNT / C ₁ sequence has a UniProtKB accession number P18640.

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of BoNT / C ₁ Clostridial toxin H _N domain.

In one embodiment modified Clostridial toxin is a BoNT / C _1, modified BoNT / C ₁ of said, 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 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid modifications); the amino acid modification (s) described above is the modified BoNT / C ₁ isoelectric point (pI) At least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) pI than otherwise identical BoNT / C ₁ pI The unit increases to a higher value. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of BoNT / D Clostridial toxin H _N domain.

  In one embodiment where the modified Clostridial toxin is BoNT / D, said 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 Including one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 62) amino acid modification; The amino acid modification (s) are at least at least isoelectric point (pI) of the modified BoNT / D, but lack the amino acid modification (s) described above and otherwise the same BoNT / B pI. 0.2 (e.g., at least 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) Increase to a higher value in pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of BoNT / E Clostridial toxin H _N domain.

  In one embodiment where the modified Clostridial 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 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 52 amino acid modifications); said amino acid modification (s) comprises the isoelectric point (pI) of the modified BoNT / E At least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) over the otherwise identical BoNT / E pI that lacks amino acid modification (s) Increase to higher value in pI units That. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of BoNT / F Clostridial toxin H _N domain.

  In one embodiment, where the modified Clostridial toxin is BoNT / F, said 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 (e.g. at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 86) Wherein the amino acid modification (s) are modified BoNT / The isoelectric point (pI) of F is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, less than the pI of the same BoNT / F, otherwise lacking the amino acid modification (s) described above. 0.6, 0.7, 0.8, 0.9, or 1) Increase to a higher value in pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of BoNT / G Clostridial toxin H _N domain.

  In one embodiment, wherein the modified Clostridial toxin is BoNT / G, said 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 (E.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 36 all) amino acid modifications; said amino acid modification (s) include modified BoNT / The isoelectric point (pI) of G is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, less than the pI of the same BoNT / G, otherwise lacking the amino acid modification (s) described above. 0.6, 0.7, 0.8, 0.9, or 1) Increase to a higher value in pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

The present inventors have identified certain amino acids corresponding to the preferred target amino acid modifications of TeNT Clostridial toxin H _N domain.

  In one embodiment where the modified Clostridial toxin is TeNT, said 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 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 49 amino acid modifications), wherein said amino acid modification (s) are the isoelectric point (pI) of the modified TeNT, otherwise lacking said amino acid modification (s) Increases to 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 a substitution of an amino acid with a lysine residue or arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  We have identified specific amino acids that represent preferred targets for amino acid modifications in the BoNT / A Clostridial toxin light chain.

  In one embodiment where the modified Clostridial toxin is BoNT / A, said 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 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 28) amino acid modifications; the amino acid modification (s) include the isoelectric point (pI) of the modified BoNT / A and the amino acid modification (s) described above. Otherwise, it increases 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 pI of the same BoNT / A. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  We have identified specific amino acids that represent preferred targets for amino acid modifications in the BoNT / B clostridial toxin light chain.

  In one embodiment, wherein the modified Clostridial toxin is BoNT / B, said 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, Including at least one amino acid modification selected from E423 and E427 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40 or 41 amino acids; The modification (s) is such that the isoelectric point (pI) of the modified BoNT / B is at least 0.2 less than the pI of the same BoNT / B otherwise lacking the amino acid modification (s) described above. (E.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) increases to a value higher by pi units.In one embodiment, the modification is performed at the lysine or arginine residue. In. One embodiment comprises substitution of Amino Acids, the modification, in. One embodiment include substitutions of amino acids with a lysine residue, wherein the modification comprises a substitution of an amino acid with arginine residues.

The inventors have identified specific amino acids that represent preferred targets for amino acid modifications in the BoNT / C ₁ clostridial toxin light chain.

In one embodiment modified Clostridial toxin is a BoNT / C _1, modified BoNT / C ₁ of said, 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, Comprising at least one amino acid selected from E427, D442 and N448 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 41 all); said amino acid modification (single) Or) has an isoelectric point (pI) of the modified BoNT / C ₁ that is at least 0.2 (pI) less than the pI of the same BoNT / C ₁ otherwise lacking the amino acid modification (s) described above. 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 a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

  In one embodiment where the modified Clostridial toxin is BoNT / D, the modified BoNT / D is D7, N9, D12, N15, D16, N17, D53, D73, D119, E124, E139, E142, N143, Q177, Q178, N180, E184, E255, N308, D335, N336, N339, N343, N368, N386, D389, D390, N391, D397, N403, N407, E409, E416 and N443 (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30 or 34) all amino acid modifications; said amino acid modification (s) is the isoelectric point (pI) of the modified BoNT / D At least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9) than the otherwise identical BoNT / D pI, which lacks the amino acid modification (s) described above. Or 1) increase to a value higher by pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

  In one embodiment where the modified Clostridial toxin is BoNT / E, said 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 Including one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or 37) amino acid modifications; said amino acid modification (s) include modified BoNT / The isoelectric point (pI) of E is at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, less than the pI of the same BoNT / E, otherwise lacking the amino acid modification (s) described above. 0.6, 0.7, 0.8, 0.9, or 1) Increase to a higher value in pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

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

  In one embodiment where the modified Clostridial toxin is BoNT / E, said modified BoNT / E is N5, N8, N10, D11, N14, D15, N72, Q75, N118, N122, Q123, N138, Q237, Q290, Q297, N362, D366, N378 and N379 selected from at least one (e.g., at least 1, 2, 3, 4, 5, 10, 15 or 19) amino acid modifications; Or) has an isoelectric point (pI) of the modified BoNT / E that is at least 0.2 (e.g., less than the pI of the same BoNT / E that otherwise lacks the amino acid modification (s) described above. 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 a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  In one embodiment where the modified Clostridial toxin is BoNT / E, said modified BoNT / E is at least one selected from N8, N10, Q75, Q123, N138 and Q237 (e.g., at least 1, 2, 3, 4, 5 or 6) amino acid modifications; wherein said amino acid modification (s) lacks the isoelectric point (pI) of the modified BoNT / E and lacks said amino acid modification (s); In other respects, it increases to 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 with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  In one embodiment, where the modified Clostridial toxin is BoNT / E, said modified BoNT / E is modified with at least one amino acid selected from Q123, N138 and Q237 (e.g., at least 1, 2, or all 3). The amino acid modification (s) include the modified BoNT / E isoelectric point (pI); otherwise, the amino acid modification (s) lack the amino acid modification (s), otherwise the same BoNT / E pI Rather than 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 a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  We have identified specific amino acids that represent preferred targets for amino acid modifications in the BoNT / F Clostridial toxin light chain.

  In one embodiment where the modified Clostridial toxin is BoNT / F, said 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 (e.g. At least 1, 2, 3, 4, 5, 10, 15, 20, 30 or 35) amino acid modifications; said amino acid modification (s) is the isoelectric point (pI) of the modified BoNT / F ) At least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, less than the pI of the same BoNT / F, otherwise lacking the amino acid modification (s) above. 0.9, or 1) Increase to a higher value by pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  We have identified specific amino acids that represent preferred targets for amino acid modifications in the BoNT / G Clostridial toxin light chain.

  In one embodiment, wherein the modified Clostridial toxin is BoNT / G, said modified BoNT / G is N4, N7, N9, N11, D12, N15, D17, E48, Q55, D57, N60, D75, D127, Q144, E148, D149, Q150, N178, E185, E208, D211, E255, D315, D332, N334, D340, E383, D387, N388, Q393, N394, E395, N403, E407, E418, E422, E426 and N443 At least one (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or 38) amino acid modifications; said amino acid modification (s) The isoelectric point (pI) of BoNT / G is at least 0.2 (e.g., at least 0.2, 0.3, 0.4) than the pI of the same BoNT / G, otherwise lacking the amino acid modification (s) described above. , 0.5, 0.6, 0.7, 0.8, 0.9, or 1) Increase to a higher value in pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

  We have identified specific amino acids that represent preferred targets for amino acid modifications in the TeNT light chain.

  In one embodiment where the modified Clostridial toxin is TeNT, said 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 (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35 or 36) all amino acid modifications; said amino acid modification (s) include the isoelectric point (pI) of the modified TeNT , Lacking the amino acid modification (s), or otherwise at least 0.2 (e.g., at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) than the same TeNT pI. ) Increases to a higher value by pI units. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue or an arginine residue. In one embodiment, the modification comprises a substitution of an amino acid with a lysine residue. In one embodiment, the modification comprises a substitution of an amino acid with an arginine residue.

The present invention is suitable for application to many different clostridial toxins. Thus, in the context of the present invention, the term “Clostridial toxin” refers to the toxin produced by C. botulinum (botulinum neurotoxin serotypes A, B, C ₁ , D, E, F and G), C Tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E) and C. baratii (botulinum neurotoxin serotype F) and modified clostridial toxins or any of the above Includes derivatives derived from crab. The term “Clostridial toxin” also encompasses 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 neurotoxins serotypes A, B, C ₁ , D, E, F, G and H, all of which have similar structures and actions 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 a percentage of sequence identity at the amino acid level. The BoNT protein of a given serotype is further divided into different subtypes based on the percentage amino acid sequence identity.

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

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

  The term “Clostridial toxin” is also intended to encompass modified Clostridial toxins and derivatives thereof including, but not limited to, those described below. A modified clostridial toxin or derivative may contain one or more amino acids that are modified compared to the natural (unmodified) form of the clostridial toxin or is not present in the natural (unmodified) form of the clostridial toxin It may contain one or more inserted amino acids. By way of example, a modified Clostridial toxin can have an amino acid sequence modified in one or more domains associated with a native (unmodified) Clostridial toxin sequence. Such modifications can modify functional aspects of the toxin, such as biological activity or persistence. Accordingly, in one embodiment, the modified Clostridial toxin of the present invention is a modified Clostridial toxin or a modified Clostridial toxin derivative or a modified Clostridial toxin derivative.

A modified Clostridial toxin can have one or more modifications in the amino acid sequence of a heavy chain (such as a modified _HC domain), wherein the modified heavy chain is associated with a target neuronal cell, Bind with higher or lower affinity than native (unmodified) clostridial 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 Clostridial toxins are described in WO2006 / 027207 and WO2006 / 114308, which are hereby incorporated by reference in their entirety.

  A modified Clostridial toxin can have one or more modifications in the amino acid sequence of the light chain, for example, 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 Clostridial toxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are hereby incorporated by reference in their entirety.

  The modified Clostridial toxin can include one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified Clostridial toxin. For example, a modified Clostridial toxin can include a leucine- or tyrosine-based motif, wherein the motif increases or decreases the biological activity and / or biological persistence of the modified Clostridial toxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM, where x is any amino acid. Suitable tyrosine-based motifs include Y-x-x-Hy, where Hy is a hydrophobic amino acid. Examples of modified Clostridial toxins containing leucine- and tyrosine-based motifs are described in WO2002 / 08268, which is incorporated herein by reference in its entirety.

  The term “Clostridial toxin” is intended to encompass hybrid and chimeric Clostridial toxins. A hybrid Clostridial toxin comprises at least a portion of a light chain from one Clostridial toxin or a subtype thereof and at least a portion of a heavy chain from another Clostridial toxin or a Clostridial toxin subtype. In one embodiment, a hybrid Clostridial toxin may contain a total light chain from one Clostridial toxin subtype and a heavy chain from another Clostridial toxin subtype. In another embodiment, a chimeric Clostridial toxin can contain a heavy chain portion (e.g., a binding domain) of certain Clostridial toxin subtypes, and another portion of the heavy chain can be transferred to another Clostridial toxin subtype. Derived from. Similarly or alternatively, the therapeutic element may comprise a light chain portion derived from a different clostridial toxin. Such hybrid or chimeric Clostridial toxins, for example, give the therapeutic benefits of such Clostridial toxins to patients who are immunologically resistant to a given Clostridial toxin subtype, Delivered to patients who may have lower than average concentrations of receptors for the chain-binding domain, or patients who may have protease-resistant variants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP and syntaxin) It is useful as a means to do this. Hybrid and chimeric Clostridial toxins are described in US 8,071,110, which is incorporated herein by reference in its entirety. Thus, in one embodiment, the modified Clostridial toxin of the present invention is a modified hybrid Clostridial toxin or a modified chimeric Clostridial toxin.

The term “Clostridial toxin” is intended to encompass retargeted Clostridial toxins. In a retargeted clostridial toxin, the clostridial toxin is modified to include an exogenous ligand known as a 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 are described, for example, in 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. Accordingly, in one embodiment, the modified Clostridial toxin of the present invention is a modified retargeted Clostridial toxin.

The present invention also includes a clostridial toxin having a non-natural protease cleavage site. In such clostridial 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 native to the clostridial toxin (i.e., an exogenous cleavage site). Replaced. Such sites require exogenous proteases for cleavage, which allows for improved control over the timing and location of the cleavage event. Non-natural protease cleavage sites that can be used in Clostridial toxins include the following:
Enterokinase (DDDDK ↓)
Factor X (IEGR ↓ / IDGR ↓)
TEV (Tobacco Etch Virus) (ENLYFQ ↓ G)
Thrombin (LVPR ↓ GS)
PreScission (LEVLFQ ↓ GP).

  Additional protease cleavage sites include recognition sequences that are cleaved by non-cytotoxic proteases, eg, by the light chain of a clostridial neurotoxin. These include SNARE (eg, SNAP-25, syntaxin, VAMP) protein recognition sequences that are cleaved by non-cytotoxic proteases such as Clostridial neurotoxin light chains. Clostridial toxins containing non-natural protease cleavage sites are described in US 7,132,259, EP 1206554-B2 and US 2007/0166332, all of which are hereby incorporated by reference in their entirety. Inteins that are self-cleaving sequences are also encompassed by the term protease cleavage site. The self-splicing reaction can be controlled, for example, by changing the concentration of reducing agent present.

  The present invention also encompasses clostridial toxins that contain “destructive cleavage sites”. In the clostridial toxin, a non-native protease cleavage site is incorporated into the clostridial toxin at selected locations such that cleavage at the site reduces or inactivates the activity of the clostridial toxin. Destructive protease cleavage sites can be sensitive to cleavage by local proteases in the event that a clostridial toxin moves to a non-target location after administration. Suitable non-natural protease cleavage sites include those described above. Clostridial toxins containing destructive cleavage sites are described in WO2010 / 094905 and WO2002 / 044199, both of which are hereby incorporated by reference in their entirety.

The modified Clostridial toxins of the invention, in particular the light chain component thereof, can be PEGylated (this can help increase stability, eg, the duration of action of the light chain component). PEGylation is particularly preferred when the light chain comprises a BoNT / A, B or C ₁ protease. PEGylation preferably involves the addition of PEG to the N-terminus of the light chain component. As an example, the N-terminus of the light chain can be extended with one or more amino acid (eg, cysteine) residues, which can be the same or different. One or more of the amino acid residues can have its own PEG molecule attached to it (eg, covalently attached). An example of this technique is described in WO2007 / 104567, which is incorporated herein by reference in its entirety.

  The modified Clostridial toxins of the present invention may not include complexing proteins present in naturally occurring Clostridial toxin complexes.

  The modified Clostridial toxins of the present 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 amino acid residues of the modified Clostridial toxin of the present invention. May be substituted. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids can be substituted for clostridial polypeptide amino acid residues. The modified Clostridial toxins of the present invention may also contain non-naturally occurring amino acid residues.

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

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

  In another aspect, the invention provides a nucleic acid (eg, DNA) comprising a nucleic acid sequence encoding a modified Clostridial toxin as described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector that includes 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 molecules of the present invention can be made using any suitable method known in the art. Thus, nucleic acid molecules can be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention can be made using molecular biology techniques.

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

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

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

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

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

  In one embodiment, the modified Clostridial toxin of the present invention is a modified BoNT / A as described above, wherein said modified BoNT / A comprises (or consists of) 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 Clostridial toxin, as described above, wherein the nucleic acid is at least 70% (e.g., for a nucleic acid sequence selected from SEQ ID NOs: 2, 4, and 6). Nucleic acid sequences having 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 Clostridial toxin of the present invention is a modified BoNT / E as described above, wherein said modified BoNT / E is at least 70% (eg, at least 75, 80, 85 relative to SEQ ID NO: 7). , 90, 95, 97, 98, 99, 99.5, or 99.9%).

  In one aspect, the invention provides a nucleic acid encoding a modified Clostridial toxin as described above, wherein the nucleic acid is at least 70% (eg, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5, or 99.9%) nucleic acid sequences having sequence identity.

  “Percent 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. Thus,% identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of% sequence identity may also take into account the number of gaps that need to be introduced to optimize the alignment of two or more sequences, the length of each gap. Sequence comparison between two or more sequences and determination of percent identity can be performed using specific methodological algorithms such as BLAST, which will be familiar to those skilled in the art.

  In one aspect, the invention provides a method for producing a single-chain modified Clostridial toxin protein having a light chain and a heavy chain, the method comprising: nucleic acid (wherein the nucleic acid is as described above) in a suitable host cell. Expressing the protein, lysing the host cell to provide a host cell homogenate containing the single chain modified Clostridial toxin protein, and isolating the single chain modified Clostridial toxin protein.

  In another aspect, the present invention provides a method for activating a modified Clostridial toxin, which method can be obtained by a method for producing a single-chain modified Clostridial toxin protein as described above. Contacting the polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the light chain and the heavy chain, whereby the polypeptide is bound to the light chain and Converting the heavy chain to a double-stranded polypeptide linked together by disulfide bonds.

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

  In related aspects, the invention provides strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, stomatognathic dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia ), Torticollis (e.g. spastic torticollis), cosmetic therapy (beauty) applications that benefit from disabling cells / muscles (by SNARE down-regulation or inactivation), neuromuscular disorders or conditions of eye movement (E.g., common strabismus, strabismus, rectus palsy, nystagmus, abnormal thyroid disease) , Spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, hatred, back spasms, leg cramps, tension headache, levator pelvic syndrome, spina bifida, tardive dyskinesia, parkinson Disease, stuttering, unilateral facial spasm, eyelid disorder, cerebral palsy, localized spasticity, spastic colitis, neurogenic bladder, anismus, extremity spasticity, tic, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis Symptom, excessive salivation, excessive gastrointestinal secretion, muscle pain (e.g. pain from muscle spasm), headache pain (e.g. tension headache), deep forehead wrinkles, fine skin wrinkles, cancer, uterine disorder, urination Provided is a modified Clostridial toxin as described above for use in the prevention or treatment of a disease or condition selected from genital disorders, genitourinary neurological disorders, chronic neurogenic inflammation and smooth muscle disorders.

  In use, the present invention uses a pharmaceutical composition comprising a modified Clostridial toxin together with at least one component selected from pharmaceutically acceptable carriers, excipients, adjuvants, propellants and / or salts. To do.

  The modified Clostridial toxins of the present invention may 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 a modified Clostridial toxin to be delivered topically, the modified Clostridial toxin may be formulated for cream (eg, for topical application) or subcutaneous injection.

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

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

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

  Dosage ranges for administration of the modified Clostridial toxins of the present invention are those that provide the desired therapeutic effect. The required dosage range will depend on the exact nature of the modified Clostridial toxin or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgment of the attending physician. It will be appreciated that it will change accordingly. These dosage level variations can be adjusted using standard experience determined methods for optimization.

  Suitable daily doses (per kg patient body weight) are 0.0001-1 ng / kg, preferably 0.0001-0.5 ng / kg, more preferably 0.002-0.5 ng / kg, particularly preferably 0.004-0.5 ng The range is / kg. Unit dosages can vary from less than 1 picogram to 30 ng, but will usually range from 0.01 to 1 ng per dose administered daily, or less frequently, such as weekly or every 6 months.

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

  Fluid dosage forms are usually prepared utilizing a modified clostridial toxin and a pyrogen-free sterile medium. The modified Clostridial toxin can be dissolved or suspended in a medium depending on the medium and concentration used. In the preparation of the solution, the modified Clostridial toxin can be dissolved in the medium, and the solution is made isotonic by the addition of sodium chloride, if necessary, and sterilized by filtration through a sterile filter using aseptic techniques. And then filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the solution stability is sufficient, the solution in the sealed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or fungicides, suspending or emulsifying agents and / or topical aesthetic agents may also be dissolved in the medium.

  Dry powders that are dissolved or suspended in a suitable medium prior to use can be prepared by filling the pre-sterilized ingredients into sterile containers using aseptic techniques in the sterile field. Alternatively, the components may be dissolved in a suitable container using aseptic techniques in the sterile field. The product is then lyophilized and the container is sealed aseptically.

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

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

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

FIG. 2 is a diagram showing SDS-PAGE purification of CatH _N _v1 (FIG. 1A), CatH _N _v2 (FIG. 1B) and CatH _N _v3 (FIG. 1C) purification. It is a figure which shows the SNAP-25 cleavage percentage in 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, and then subjected to Western blot using SNAP-25 specific antibody. Data are mean ± SEM from triplicate independent experiments. FIG. 5 shows the efficacy (t ₅₀ ) of BoNT / A and CatH _N _v1 in the mouse phrenic nerve unilateral diaphragm assay (mPNHD). Data points are individual unilateral diaphragm preparations and mean ± SEM. CatH _{N —} v1 was statistically significantly slower than the reference protein BoNT / A (List Biological Laboratories). One-way analysis of variance and Dunnett's multiple comparison test. ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 (one-way analysis of variance and Dunnett's multiple comparison test). It is a figure which shows the SNAP-25 cutting | disconnection percentage in 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, and then Western blotted with SNAP-25 specific antibodies. Data are mean ± SEM from triplicate independent experiments. FIG. 6 shows the efficacy (t ₅₀ ) of BoNT / A and CatH _{N —} v2 in the mouse phrenic nerve hemidiaphragm assay (mPNHD). Data points are individual unilateral diaphragm preparations and mean ± SEM. CatH _N _v2 was statistically equivalent to the reference protein BoNT / A (List Biological Laboratories). One-way analysis of variance and Dunnett's multiple comparison test. ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 (one-way analysis of variance and Dunnett's multiple comparison test). It is a figure which shows the SNAP-25 cleavage 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, and then Western blotted with SNAP-25 specific antibodies. Data are mean ± SEM from triplicate independent experiments. FIG. 4 shows the efficacy (t ₅₀ ) of BoNT / A and CatH _N _v3 in the mouse phrenic nerve unilateral diaphragm assay (mPNHD). Data points are individual unilateral diaphragm preparations and mean ± SEM. CatH _{N —} v3 was statistically significantly slower than the reference protein BoNT / A (List Biological Laboratories). One-way analysis of variance and Dunnett's multiple comparison test. ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 (one-way analysis of variance and Dunnett's multiple comparison test). It is a figure which shows an isoelectric focusing electrophoresis analysis. All three CatH _N constructs have an observed increase in pI compared to unmodified BoNT / A. It is a figure which shows SDS-PAGE refinement | purification of a CatLC construct. CatLC catalytic activity, pEC ₅₀ value compared to BoNT / E LC reference was obtained in the BoTest A / E BoNT detection kit (BioSentinal catalog number A1004) according to the manufacturer's instructions. Data show the mean ± standard deviation obtained from one independent experiment in triplicate.

  Array

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 certain embodiments of the invention and do not in any way limit the scope of the invention as defined in the claims.

  [Example 1]

  Identification of preferred clostridial 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. Position related to 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 AreaIMol and the exposed residues were identified as having a total value greater than 55.

The secondary structure of the H _N and TeNT for each subtype were identified using the secondary structure assignment program (Stride Web Interface). Regions assigned to form an α-helix, β chain or 3 ₁₀ -helix were excluded from 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.

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

BoNT / A:
D474, N476, D484, N486, I487, E488, A489, A490, E491, D546, E558, E560, H561, I566, L568, N570, S571, L577, N578, A597, E599, A601, E620, V621, T623, D625, T631, N645, L647, D650, D651, I668, E670, A672, V675, S683, I685, A686, N687, N752, Q753, T755, 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 clostridial toxins for modification in the BoNT / E light chain amino acid residues:
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 molecules

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

Using the method described in Example 1, a total of 55 residues were identified as candidates for mutation in the BoNT / AH _N domain. Residue compatibility was further assessed by visual inspection of the BoNT / A crystal structure, and 11 preferred candidates (N476, N763, N687, E599, I831, N761, N578, V675, I685, T755, E757) A list was obtained. An additional 6 residues were selected based on functional data indicating that these residues were able to accept mutations without adversely affecting protein function. Of these residues, 4 were in the candidate list (L647, D650, D651, T847) and 2 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 selected 17 residues: CatH _N _v1, CatH _N _v2 and CatH _N _v3. CatH _N construct mutations are shown in Table 3 below:

Purification of CatH _N _v1, CatH _N _v2 and CatH _N _v3 is shown in FIGS. 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 the pJ401 expression vector, and then transformed into BL21 (DE3) E. coli. This allowed soluble overexpression of recombinant modified BoNT / A molecules in E. coli.

  Recombinant modified BoNT was purified from E. coli lysates using traditional chromatographic techniques. An initial purification step using a cation exchange resin was used followed by an intermediate purification step using a hydrophobic interaction resin. The recombinant modified BoNT single chain was then cleaved by proteolysis, resulting in an activated double-stranded modified BoNT. A final purification step was then used to remove residual contaminants.

  [Example 4]

  Characterization of purified modified BoNT

  The modified BoNT described in Example 2 above was characterized by experiment.

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

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

The CatH _N _v1 molecule was tested in a rat embryonic spinal 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 (FIG. 3).

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

CatH _N _v2 protein was tested in an eSCN SNAP-25 cleavage assay and found to retain the ability to enter cells and cleave SNAP-25. In the mPNHD assay, CatH _N _v2 was equipotent with BoNT / A (FIGS. 4 and 5).

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

CatH _N _v3 molecules were tested in the eSCN SNAP-25 cleavage assay and found to retain the ability to enter cells and cleave SNAP-25. Similarly, positive results were demonstrated in the mPNHD assay (FIGS. 6 and 7).

  Isoelectric focusing

All three CatH _N constructs have an increased pI compared to unmodified BoNT / A (FIG. 8).

  [Example 5]

  BoNT / E light chain modification

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

  BoNT / E light chain constructs (“CatLC”) with the mutations shown in the table below were prepared.

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

  Evaluation of the construct for catalytic activity indicated that the modified light chain retains the catalytic activity of the unmodified light chain (FIG. 10).

item
1. A modified Clostridial toxin comprising at least one amino acid modification, wherein the at least one amino acid modification is the isoelectric point (pI) of the modified Clostridial 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 clostridial toxin,
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 Clostridial toxin according to Item 1, wherein the at least one amino acid modification is located in a Clostridial toxin translocation domain (H _N domain).
3. The at least one amino acid modification is located in a clostridial toxin light chain,
The modified clostridial toxin according to item 1.
4. The modified Clostridial toxin according to Item 3, wherein the at least one amino acid modification does not introduce an E3 ligase recognition motif into the Clostridial toxin light chain.
5. The at least one amino acid modification has an isoelectric point (pI) of the modified Clostridial toxin that is at least 0.5 pI less than the pI of the other Clostridial toxin that otherwise lacks the at least one amino acid modification. 5. The modified Clostridial toxin according to any one of Items 1 to 4, which increases to a unit high value.
6. The at least one amino acid modification has an isoelectric point (pI) of the modified Clostridial toxin that is at least 1 pl unit greater than the pI of the other Clostridial toxin that otherwise lacks the at least one amino acid modification. 5. The modified Clostridial toxin according to any one of Items 1 to 4, which increases to a high value.
7. The at least one amino acid modification has an isoelectric point (pI) of the modified Clostridial toxin, which is at least 2 pI units greater than the pI of the other Clostridial toxin otherwise lacking the at least one amino acid modification. 5. The modified Clostridial toxin according to any one of Items 1 to 4, which increases to a high value.
8. The at least one amino acid modification is 2 to 5 greater than the isoelectric point (pI) of the modified Clostridial toxin than the pI of the otherwise identical Clostridial toxin that lacks the at least one amino acid modification. 5. The modified Clostridial toxin according to any one of Items 1 to 4, which increases to a high pI unit value between
9. A modified Clostridial toxin according to any of the preceding items having a pI of at least 6.5.
10. The modified Clostridial toxin according to any of the preceding items, having a pI between 6.5 and 9.5.
11. The modified Clostridial toxin according to any of the preceding items, having a pI between 6.5 and 7.5.
12. The modified Clostridial toxin according to any of the preceding items, wherein the 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 Clostridial toxin according to Item 12, wherein
14. The modified Clostridial toxin according to any of the preceding items, comprising between 1 and 90 amino acid modifications.
15. The modified Clostridial toxin according to any of the preceding items comprising at least three amino acid modifications.
16. The modified Clostridial toxin according to any of the preceding items, comprising between 4 and 40 amino acid modifications.
17. The modified Clostridial toxin according to any of the preceding items, wherein the at least one amino acid modification is a modification of a surface exposed amino acid residue.
18. At least one amino acid modification is an aspartic acid residue, glutamic acid residue, histidine residue, asparagine residue, glutamine residue, serine residue, threonine residue, alanine residue, glycine residue, valine residue, The modified Clostridial toxin according to any of the preceding items, comprising a modification of an amino acid residue selected from leucine residues and isoleucine residues.
19. The modified Clostridial toxin according to Item 18, wherein the amino acid residue is substituted with a lysine residue or an arginine residue.
20. A nucleic acid comprising a nucleic acid sequence encoding a modified Clostridial toxin according to any one of items 1 to 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 produce a single-chain modified Clostridial toxin protein. Providing a host cell homogenate containing the chain-modified clostridial toxin protein and isolating the single-chain modified clostridial toxin protein.
22. A method of activating a modified Clostridial toxin, comprising providing a single-chain modified Clostridial toxin protein obtained by the method of Item 21, and wherein the polypeptide is between a light chain and a heavy chain. Contacting a protease that cleaves at a located recognition site (cleavage site) and converting the polypeptide to a double-stranded polypeptide in which the light and heavy chains are linked together by disulfide bonds. Method.
23. The modified Clostridial toxin according to any one of Items 1 to 19, for use in medicine.
24.strabismus, blepharospasm, squint, dystonia (eg spastic dystonia, stomatognathic dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia) , Spastic torticollis), cosmetic therapy (beauty) applications that benefit from disabling cells / muscles (by SNARE down-regulation or inactivation), neuromuscular disorders or conditions of eye movement (e.g., joint strabismus) (concomitant strabismus), vertical strabismus, rectus palsy, nystagmus, thyroid abnormal muscle disease), writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tic, segmental myoclonus, convulsions, chronic polymorphism Spasticity due to sclerosis, spasticity resulting in abnormal bladder control, hatred, back spasms, leg spasms, tension headache, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering Unilateral facial spasm, eyelid disorder, cerebral palsy, focal spasticity, spastic colitis, neural bladder, anismus, extremity spasm, tic, tremor, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive Saliva secretion, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache), deep wrinkles, fine wrinkles on the skin, cancer, uterine disorders, genitourinary disorders, 20. The modified Clostridial toxin according to any one of Items 1 to 19, for use in the prevention or treatment of a disease or condition selected from urogenital neurological disease, chronic neurogenic inflammation and smooth muscle disorder.
25. A modified Clostridial toxin with reference to the accompanying drawings substantially as described herein.

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

Claims (1)

A modified Clostridial toxin comprising at least one amino acid modification, wherein the at least one amino acid modification is the isoelectric point (pI) of the modified Clostridial toxin, otherwise lacking the at least one amino acid modification, Increased to at least 0.2 pI units higher than the pI of the same clostridial toxin,
Wherein the at least one amino acid modification, not located in a Clostridial toxin binding domain (H _C domain), a modified Clostridial toxin.