NZ715570B2 - Cationic neurotoxins - Google Patents
Cationic neurotoxins Download PDFInfo
- Publication number
- NZ715570B2 NZ715570B2 NZ715570A NZ71557014A NZ715570B2 NZ 715570 B2 NZ715570 B2 NZ 715570B2 NZ 715570 A NZ715570 A NZ 715570A NZ 71557014 A NZ71557014 A NZ 71557014A NZ 715570 B2 NZ715570 B2 NZ 715570B2
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- New Zealand
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- asn
- asp
- amino acid
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- clostridial toxin
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Classifications
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- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
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- C12Y—ENZYMES
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- C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
Abstract
The present invention provides an engineered clostridial toxin comprising at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification. Also provided are corresponding uses of the engineered clostridial toxin in therapy. otherwise identical clostridial toxin lacking said at least one amino acid modification. Also provided are corresponding uses of the engineered clostridial toxin in therapy.
Description
CATIONIC NEUROTOXINS The present invention relates to engineered clostridial toxins comprising at least one amino acid modification, and the use of such engineered clostridial toxins in medicine and therapy.
Bacteria in the genus Clostridia e highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such idial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, as well as those produced by C. baratii and C. butyricum.
Among the idial toxins are some of the most potent toxins known. By way of example, botulinum oxins have median lethal dose (LD50) values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype. Both tetanus and botulinum toxins act by inhibiting the on of affected neurons, specifically the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central s system.
In nature, clostridial toxins are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. ge occurs at a specific cleavage site, often referred to as the activation site, that is located between the cysteine residues that provide the inter-chain disulphide bond. It is this di-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H-chain), which has a lar mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of imately 50 kDa. The H-chain comprises an N-terminal translocation component (HN domain) and a C-terminal ing component (He domain). The cleavage site is located between the L-chain and the translocation domain components. Following binding of the ¾ domain to its target neuron and internalisation of the bound toxin into the cell via an endosome, the HN domain translocates the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a totoxic protease).
[Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by r ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar Non-cytotoxic ses act by proteolytically cleaving intracellular transport ns known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin) — see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term e NSF Attachment kceptor, where NSF means N-ethylmaleimide-Sensitive Eactor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell. The protease fimction is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins. Accordingly, once delivered to a desired target cell, the non—cytotoxic protease is capable of inhibiting cellular secretion from the target cell. The L-chain proteases of clostridial toxins are non- cytotoxic proteases that cleave SNARE proteins.
In view of the ubiquitous nature of SNARE proteins, clostridial toxins such as botulinum toxin have been successfully employed in a wide range of therapies.
By way of example, we refer to William J. Lipham, Cosmetic and al Applications of num Toxin (Slack, Inc., 2004), which describes the use of clostridial , such as botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and tetanus neurotoxin (TeNT), to inhibit neuronal transmission in a wide variety of therapeutic and cosmetic applications - as an example, BOTOXTM is currently approved as a therapeutic for the following indications: achalasia, adult spasticity, anal fissure, back pain, blepharospasm, bruxism, cervical ia, essential tremor, glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, ctivity of bladder, hyperhidrosis, le cerebral palsy, multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismus and VII nerve disorder. In addition, clostridial toxin therapies are described for treating neuromuscular disorders (see US 6,872,397), for treating uterine disorders (see US 2004/0175399); for treating ulcers and esophageal reflux disease (see US 2004/0086531), for treating dystonia (see US 505); for treating eye disorders (see US 2004/0234532), for treating blepharospasm (see US 2004/0151740), for treating strabismus (see US 2004/0126396), for ng pain (see US 6,869,610, US 6,641,820, US 6,464,986, and US 6,113,915), for ng fibromyalgia (see US 742, US 2004/0062776); fneating lower back pain (see US 2004/0037852), for treating muscle injuries (see [Annotation] wilksar None set by wilksar [Annotation] r ionNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar US 6,423,319), for treating sinus he (see US 6,838,434); for treating tension headache (see US 6,776,992); for treating headache (see US 6,458,365); for reduction of migraine headache pain (see US 5,714,469); for treating vascular diseases (see US 6,767,544); for treating neurological disorders such as Parkinson's disease (see US 6,620,415, US 6,306,403); for treating neuropsychiatric disorders (see US 2004/0180061, US 2003/0211121), for treating endocrine disorders (see US 6,827,931), for treating thyroid disorders (see US 6,740,321); for ng cholinergic influenced sweat gland disorders (see US 6,683,049); for treating diabetes (see US 6,337,075, US 6,416,765), for treating a pancreatic disorder (see US 6,261,572, US 6,143,306), for treating cancers such as bone tumors (see US 6,565,870, US 6,368,605, US 6,139,845, US 2005/0031648); for treating otic ers (see US 926, US 379), for treating autonomic disorders such as gastrointestinal muscle disorders and other smooth muscle dysfunction (see US 5,437,291); for treatment of skin lesions associated with cutaneous cell-proliferative disorders (see US 484); for management of neurogenic inflammatory disorders (see US 6,063,768), for reducing hair loss and stimulating hair growth (see US 6,299,893), for treating downturned mouth (see US 6,358,917); for reducing appetite (see US 2004/40253274), for dental ies and procedures (see US 2004/0115139); for treating neuromuscular disorders and conditions (see US 2002/0010138), for treating various disorders and conditions and associated pain (see US 2004/0013692); for treating conditions resulting from mucus hypersecretion such as asthma and COPD (see W0 00/10598); and for treating non-neuronal conditions such as inflammation, endocrine conditions, exocrine conditions, immunological conditions, vascular conditions, bone conditions (see WO 01/21213). All of the above publications are hereby incorporated by reference in their entirety.
The use of non-cytotoxic ses such as clostridial toxins (e. g. BoNTs and TeNT) in eutic and cosmetic treatments of humans and other mammals is pated to expand to an ever-widening range of diseases and ts that can benefit from the properties of these toxins.
To avoid systemic neurological effects, many clostridial toxin therapies utilise direct administration of the idial toxin therapeutic to a given target site (such as a tgt tissue). A problem when administering clostridial toxin—based therapeutics in [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar this fashion is the spread of toxin away from the administration site and into surrounding tissue or systemic circulation. The diffusion of toxin away from the target tissue is believed to be responsible for undesirable side effects that in extreme cases may be life threatening. This can be a particular concern when using clostridial toxin therapeutics (such as BoNT therapeutics) at high doses, trations and injection s. Adverse effects associated with this m that have been reported for commercial BoNT/A therapeutics include ia, generalised muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and breathing difficulties. Swallowing and ing difficulties can be life threatening and there have been reported deaths related to the spread of toxin effects.
There is therefore a need in the art for clostridial toxins which have properties of increased tissue retention at the site of administration, and which accordingly exhibit a reduction in diffusion away from the administration site, as compared to known clostridial toxins.
The present invention solves the above problem by ing engineered clostridial toxins, as specified in the claims.
In one aspect, the invention es an engineered clostridial toxin comprising at least one amino acid modification, wherein said at least one amino acid modification increases the ctric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one ment, said at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 0.4 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one embodiment, said at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 05 p1 units higher than the pI of an otherwise cal clostridial toxin lacking said at least one amino acid modification. In one embodiment, said at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 0.6 pI units higher than the pI of an otherwise identical clostridial tn lacking said at least one amino acid ation. In one embodiment, said at [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 0.8 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one embodiment, said at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 1 p1 unit higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
In one aspect, the invention es an engineered idial toxin comprising at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least one pI unit higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
In certain embodiments, the engineered clostridial toxin comprises at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 amino acid modifications.
In certain embodiments, said at least one amino acid modification increases the pI of the engineered clostridial toxin to a value that is at least 2, 3, 4 or 5 p1 units higher than the pI of an otherwise cal clostridial toxin lacking said at least one amino acid ation.
The t ors have found that by increasing the pI of a clostridial toxin, for example, by at least 02 p1 units, or 0.5 pI units, or one pI unit (through the introduction into the clostridial toxin protein of at least one amino acid modification), the resultant engineered clostridial toxin ageously demonstrates properties of increased tissue retention and reduced diffusion away from sites of administration, while retaining abilities of target cell binding, translocation, and cleavage of target SNARE protein(s). Thus, the spread of clostridial toxin from the site of administration is cantly reduced, as compared to an ise identical clostridial toxin lacking said at least one amino acid modification.
The engineered clostridial toxins of the invention are le for use in any of the therapies described above, and ageously may demonstrate a reduction in, or ance of, side effects compared to the use of known clostridial toxin therapeutics.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r The increased tissue retention properties of the engineered clostridial toxins of the invention also provide increased potency and/or on of action, and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional e effects), thus providing further ages.
As discussed below in more detail, the increase in pI provided by the at least one amino acid modification means that an engineered clostridial toxin of the invention has, at a given pH, a net charge that is more positive than the net charge on an otherwise identical idial toxin lacking said at least one amino acid modification.
Without wishing to be bound by any one theory, the present inventors believe that this increased positive charge allows the engineered clostridial toxins of the present invention to display longer tissue retention times at the site of administration due to favourable electrostatic interactions between the engineered clostridial toxin and anionic extracellular components (such as cell membranes and heparin sulphate proteoglycans) at the site of administration. These ed electrostatic interactions serve to reduce the diffusion of the engineered clostridial toxin away from the site of administration, thus improving tissue retention.
By way of example, the improved tissue retention properties of an engineered idial toxin of the invention may allow for (i) higher doses into dual muscles, such as the sternocleidomastoid, without spreading into nearby muscles in the neck to cause difficult swallowing, and (ii) higher total doses (to all muscles) in a single treatment, without spreading into the circulation and g systemic effects such as difficult breathing. Advantages to patients may include more effective treatment of large muscles such as the cleidomastoid muscle, increased opportunity to inject several different s during each ent, and possible longer duration of effective treatment r before re-treatment is necessary) because of higher dosing.
In one embodiment, an engineered idial toxin of the invention has, in use, a positive net charge (for example, when the engineered clostridial toxin, in use, is located at a desired administration site in a tissue).
[Annotation] r None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar The isoelectric point (p1) is a specific property of a given protein. As is well known in the art, proteins are made from a specific sequence of amino acids (also referred to when in a protein as amino acid es). Each amino acid of the standard set of twenty has a different side chain (or R group), meaning that each amino acid residue in a protein displays different chemical properties such as charge and hobicity.
These properties may be ced by the surrounding al environment, such as the ature and pH. The overall chemical teristics of a protein will depend on the sum of these various s.
Certain amino acid residues (detailed below) possess ionisable side chains that may display an electric charge depending on the surrounding pH. Whether such a side chain is charged or not at a given pH depends on the pKa of the nt ionisable moiety, wherein pKa is the negative logarithm of the acid dissociation constant (Ka) for a specified proton from a conjugate base.
For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values of approximately 4.1 (precise pKa values may depend on temperature, ionic th and the microenvironment of the ionisable group). Thus, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as "physiological pH"). At low pH values, these side chains will become protonated and lose their charge.
Conversely, basic residues such as lysine and arginine have nitrogen—containing side chain groups with pKa values of approximately 10-12. These side chains therefore exhibit a positive charge at a pH of 7.4. These side chains will become de-protonated and lose their charge at high pH values.
The l (net) charge of a protein molecule therefore depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and on the surrounding pH. Changing the surrounding pH changes the overall charge on the protein. Accordingly, for every protein there is a given pH at which the number of positive and negative charges is equal and the protein displays no overall net charfe. This point is known as the isoelectric point (p1). The isoelectric point is a [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar standard concept in n biochemistry with which the skilled person would be familiar.
The isoelectric point (p1) is ore defined as the pH value at which a protein displays a net charge of zero. An increase in pI means that a higher pH value is required for the protein to display a net charge of zero. Thus, an increase in p1 represents an increase in the net positive charge of a protein at a given pH.
Conversely, a decrease in pI means that a lower pH value is required for the protein to y a net charge of zero. Thus, a decrease in pI represents a decrease in the net positive charge of a protein at a given pH.
Methods of determining the pI of a protein are known in the art and would be familiar to a skilled person. By way of example, the pI of a protein can be calculated from the average pKa values of each amino acid present in the n. Alternatively, the pI of a protein can be determined experimentally using the technique of isoelectric ng. This technique uses electrophoresis to separate proteins according to their pI. ctric focusing is typically performed using a gel that has an immobilised pH nt. When an ic field is applied, the protein es through the pH gradient until it reaches the pH at which it has zero net charge, this point being the pI ofthe protein.
The pI of a n may be sed or decreased by altering the number of basic and/or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the n. For example, an increase in p1 may 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.
Native (unmodified) clostridial toxins have a pI of approximately 5—6. Thus, at a pH of 7.4, native botulinum toxins possess a negative net charge. By way of example, the pI ofBoNT/A is 6.4, and a BoNT/A molecule has a net charge at pH 7.4 of —8. These p1 values are calculated as described above.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar ation] wilksar Unmarked set by r TABLE 1 : CLOSTRIDIAL TOXIN pI Bo\T/A 6.4 BoRT/B 5.3 Bo\T/C1 5.5 BoRT/D 5.5 Bo\T/E 6.0 BoRT/F 5.6 G 5.2 TeNT 5.8 As described above, in one embodiment, an engineered clostridial toxin of the t invention comprises at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of an otherwise cal clostridial toxin lacking said at least one amino acid modification.
Thus, in the context of the present invention, an increase in p1 of 0.2 units in the context of an engineered BoNT/A clostridial toxin would be an increase in pI from 6.4 to 6.6.
As described above, in one embodiment, an engineered clostridial toxin of the present invention comprises at least one amino acid modification, wherein said at least one amino acid ation increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least one pI unit higher than the p1 of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
Thus, in the context of the present invention, an increase in pI of 1 unit in the context of an engineered BoNT/A clostridial toxin would be an increase in pI from 6.4 to 7.4.
In one embodiment, said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least two pI units [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by r higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
In one ment, said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is between 2 and 5 p1 units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
In one ment, the engineered clostridial toxin has a pI of at least 6 (for example, at least 6, at least 7, at least 8, or at least 9).
In one embodiment, the engineered clostridial toxin has a pI of at least 7.
In one embodiment, the engineered clostridial toxin has a pI of between 6 and 10 (for example a pI of between 7 and 9, or a pI of between 8 and 9).
As discussed above, the engineered clostridial toxins of the present invention have increased tissue retention properties that also provide increased potency and/or duration of action, and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional effects).
One way in which these advantageous ties (which represent an increase in the therapeutic index) may be defined is in terms of the Safety Ratio of the engineered clostridial toxin. In this regard, undesired effects of a clostridial toxin (caused by diffusion of the toxin away from the site of administration) can be assessed experimentally by measuring percentage bodyweight loss in a relevant animal model (eg. a mouse, where loss of bodyweight is detected within seven days of administration). Conversely, desired on-target effects of a clostridial toxin can be assessed experimentally by Digital Abduction Score (DAS) assay, a measurement of muscle paralysis. The DAS assay may be performed by injection of 20 pl of idial toxin, formulated in Gelatin Phosphate Buffer, into the mouse cnemius/soleus complex, ed by ment of Digital Abduction Score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820, 2001). In the DAS assay, mice are tsawjnded briefly by the tail in order to elicit a characteristic startle response in whichouse s its hind limbs and abducts its hind digits. ing clostridial [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar toxin injection, the varying degrees of digit abduction are scored on a five-point scale (O=normal to 4=maximal reduction in digit abduction and leg extension).
The Safety Ratio of a clostridial toxin may then be expressed as the ratio n the amount of toxin required for a 10% drop in a bodyweight (measured at peak effect within the first seven days after dosing in a mouse) and the amount of toxin ed for a DAS score of 2. High Safety Ratio scores are therefore desired, and indicate a toxin that is able to effectively se a target muscle with little undesired off-target s. An engineered toxin of the present invention has a Safety Ratio that is higher than the Safety Ratio of an equivalent unmodified (native) botulinum toxin.
Thus, in one embodiment, an engineered clostridial toxin of the present invention has a Safety Ratio of at least 8 (for example, at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50), wherein Safety Ratio is calculated as: dose of toxin required for -10% bodyweight change (pg/mouse) divided by DAS ED50 (pg/mouse) [ED50 = dose ed to produce a DAS score of 2].
In one embodiment, an engineered idial toxin of the present invention has a Safety Ratio of at least 10. In one ment, an engineered idial toxin of the present invention has a Safety Ratio of at least 15.
An engineered clostridial toxin of the present invention comprises at least one amino acid modification. Said at least one amino acid modification increases the pI of the clostridial toxin, as discussed above. In the context of the present invention, an amino acid modification is a modification of the amino acid sequence of a clostridial toxin.
Such a modification may be effected by replacing one amino acid in the ce with another (i.e. a substitution), by inserting a new amino acid into the sequence, or by deleting an amino acid of the sequence. Amino acids incorporated into an amino acid sequence in a protein are also referred to as amino acid es.
The 20 standard amino acids found in proteins are as follows: [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r ionNone set by wilksar [Annotation] r Unmarked set by wilksar TABLE 2: AMINO ACID SIDE CHAIN Aspartic acid Asp D Charged (acidic) ic acid Glu E Charged (acidic) Arginine Arg R Charged (basic) Lysine Lys K Charged (basic) Histidine His H Uncharged (polar) Asparagine Asn N Uncharged (polar) Glutamine Gln Q Uncharged (polar) Serine Ser S ged (polar) Threonine Thr T Uncharged (polar) Tyrosine Tyr Y Uncharged (polar) Methionine Met M Uncharged (polar) Tryptophan Trp W Uncharged (polar) Cysteine Cys C Uncharged (polar) Alanine Ala A Uncharged (hydrophobic) Glycine Gly G Uncharged (hydrophobic) Valine Val V Uncharged phobic) e Leu L ged (hydrophobic) Isoleucine Ile I Uncharged (hydrophobic) Proline Pro P ged (hydrophobic) Phenylalanine Phe F Uncharged (hydrophobic) 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 ic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa .8) have a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar The following amino acids are considered uncharged, polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, ne, methionine, tryptophan.
The following amino acids are considered uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.
An se in the pl of a idial toxin can be effected by introducing into the clostridial toxin one or more amino acid modifications that increases the ratio of positive to negative charges in the clostridial toxin.
In one embodiment, the at least one amino acid modification is selected from: an amino acid substitution, an amino acid insertion, and an amino acid deletion.
In an amino acid substitution, an amino acid residue that forms part of the clostridial toxin amino acid sequence is replaced with a different amino acid residue. The replacement amino acid e may be one of the 20 rd amino acids, as described above.
Alternatively, the replacement amino acid in an amino acid substitution may be a non- standard amino acid (an amino acid that is not part of the standard set of 20 described above). By way of example, the replacement amino acid may be a basic andard amino acid, e.g. L-Omithine, L—2—aminoguanidinopropionic acid, or D-isomers of Lysine, Arginine and Omithine). Methods for introducing non-standard amino acids into proteins are known in the art, and e recombinant protein synthesis using E. coli auxotrophic expression hosts.
In an amino acid insertion, an additional amino acid residue (one that is not ly present) is incorporated into the clostridial toxin amino acid ce, thus increasing the total number of amino acid residues in said sequence. In an amino acid deletion, an amino acid residue is removed from the clostridial toxin amino acid ce, thus reducing the total number of amino acid residues in said sequence.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar ed set by wilksar Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. By way of example, amino acid modifications may be introduced by modification of a DNA sequence encoding a clostridial toxin. This can be achieved using standard molecular cloning techniques, for example by irected mutagenesis where short strands of DNA (oligonucleotides) coding for the desired amino acid(s) are used to replace the original coding sequence using a polymerase , or by inserting/deleting parts of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively a modified gene sequence can be ally synthesised.
In one embodiment, the at least one amino acid modification is selected from: substitution of an acidic amino acid residue with a basic amino acid residue; substitution of an acidic amino acid residue with an ged amino acid residue; substitution of an uncharged amino acid e with a basic amino acid residue; insertion of a basic amino acid residue, and deletion of an acidic amino acid residue.
In a preferred embodiment, the at least one amino acid modification is a tution, which advantageously 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 e with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, the basic amino acid residue is a lysine residue or an arginine residue. In one embodiment, the basic amino acid residue is a lysine residue. In one embodiment, the basic amino acid residue is an arginine residue. In one ment, wherein 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 e (i.e. aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine), An engineered idial toxin of the invention may comprise more than one amino acid modification. Thus, in one embodiment, the engineered idial toxin (as described above) comprises between 1 and 80 amino acid modifications (for example, baeen 1 and 70, between 1 and 60, between 1 and 50, between 4 and 40, between 4 [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar and 30, between 5 and 40, between 5 and 30, or between 10 and 25 amino acid modifications). In one embodiment, the engineered idial toxin (as bed above) comprises between 4 and 40 amino acid modifications. In one ment, the engineered clostridial toxin comprises at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid modifications. In one embodiment, the engineered clostridial toxin comprises at least 4 amino acid modifications (for example, at least 4 amino acid substitutions). Each of said amino acid modifications is an amino acid modification as described above. Thus, each of said amino acid modifications contributes to the se in pI of the ered clostridial toxin (as compared to the pI of an otherwise cal clostridial toxin lacking said amino acid modifications).
Any clostridial toxin amino acid (i.e. amino acid residue) can be modified as described above, as long as the outcome of said modification is an increase in the idial toxin pI (as described above). However, the present inventors have identified subsets of clostridial toxin amino acids that are ularly suitable targets for modification.
Preferred target amino acids may possess certain qualities. By way of example, a preferred target amino acid may be: (i) a surface exposed amino acid; (ii) located outside of a clostridial toxin protein secondary structure; (iii) located in a clostridial toxin protein region that is non-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 a predicted ubiquitination site; or (v) any combination of the foregoing.
As discussed above, clostridial toxins are formed from two polypeptide chains, the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises a C-terminal ing component (receptor binding domain or Hc domain) and an N—terminal ocation component (HN ).
In one embodiment, the at least one amino acid modification (as described above) is located in the clostridial toxin receptor binding domain (Hc domain).
[Annotation] wilksar None set by r [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Examples of light chain reference sequences include: Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440 Botulinum type C1 oxin: 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 es 1-439 num type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457 The above-identified reference sequences should be considered a guide, as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference in its entirety) cites slightly different clostridial sequences: Botulinum type A neurotoxin: amino acid residues Ml-K448 Botulinum type B neurotoxin: amino acid residues Ml—K44l Botulinum type C1 neurotoxin: amino acid residues Ml-K449 num type D neurotoxin: amino acid residues Ml-R445 Botulinum type E neurotoxin: amino acid residues Ml-R422 Botulinum type F oxin: amino acid residues Ml-K439 Botulinum type G neurotoxin: amino acid residues Ml-K446 Tetanus neurotoxin: amino acid residues Ml-A457 es of clostridial toxin Hc domain reference sequences include: Bo\T/A - N872—L1296 B - E859-E129l 1- N867-E129l Bo\'T/D - SS63-E1276 Bo\T/E - R846-K1252 BoVT/F - K865-E1274 Bo\T/G - N864-E1297 TeNT Dl315 [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar The Hc domain of a clostridial toxin (such as a BoNT) comprises two distinct structural features that are referred to as the Hcc and HCN s. Amino acid residues ed in receptor binding are believed to be primarily located in the Hcc domain.
In one embodiment, wherein the at least one amino acid modification (as described above) is located in the clostridial toxin receptor binding domain (Hc domain), said at least one amino acid modification is located in the clostridial toxin HCN domain (also ed to as a translocation facilitating domain). In one ment, wherein the at least one amino acid modification (as described above) is located in the clostridial toxin receptor binding domain (Hc domain), said at least one amino acid ation is located in the clostridial toxin Hcc domain.
Examples of clostridial toxin HCN domain reference ces include: Botulinum type A neurotoxin: amino acid residues 872-1110 num type B neurotoxin: amino acid residues 859-1097 Botulinum type C1 neurotoxin: amino acid residues 11 Botulinum type D neurotoxin: amino acid residues 863-1098 num type B neurotoxin: amino acid residues 846-1085 Botulinum type F neurotoxin: amino acid residues 05 Botulinum type G neurotoxin: amino acid residues 864-1105 Tetanus oxin: amino acid residues 880-1127 The above sequence positions may vary slightly according to serotype/sub-type, and r examples of suitable (reference) clostridial toxin HCN domains include: Botulinum type A oxin: amino acid residues 874—1110 Botulinum type B neurotoxin: amino acid residues 861-1097 Botulinum type C1 neurotoxin: amino acid residues 869-1111 Botulinum type D neurotoxin: amino acid residues 865-1098 Botulinum type E neurotoxin: amino acid residues 848-1085 Botulinum type F neurotoxin: amino acid residues 867-1105 Botulinum type G neurotoxin: amino acid residues 866-1105 Tetanus neurotoxin: amino acid residues 882-1127 [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by r [Annotation] wilksar Unmarked set by r 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 es are those t on the exterior of a folded protein and so accessible to the surrounding solvent, in contrast to those amino acid residues that are located in the or of a folded protein. The degree of surface exposure of an amino acid residue and thus its exposure to the nding solvent depends on its position within the folded protein, and also on the conformation adopted by the protein. ation of an amino acid residue with a high degree of surface re may therefore have a greater effect on the protein’s isoelectric point than modification of an amino acid residue with a low degree of surface exposure. Methods for determining the degree of surface exposure of an amino acid residue are known in the art. By way of example, the computer program AreaIMol (part of the CCP4 suite of computer programs) can be used to calculate the degree of surface exposure of amino acid residues in a given protein. Surface exposed amino acid residues may also be identified by visual inspection of a protein crystal structure (such as provided by X—ray crystallography).
In one embodiment, a surface exposed amino acid residue has a sum AreaIMol value of at least 40.
In one embodiment, the at least one amino acid modification comprises modification of an amino acid residue selected from: an aspartic acid residue, a glutamic acid residue, a ine e, a serine e, a threonine residue, an asparagine residue, a glutamine residue, a cysteine residue, or a tyrosine residue. The present inventors have fied that amino acid residues from this group (negatively charged residues and polar es) represent particularly suitable targets for modification according to the present invention. Without wishing to be bound by any one theory, the present inventors believe that amino acid residues in this group appear on the surface of a clostridial toxin with a greater frequency than the hydrophobic residues not listed.
In one embodiment, wherein the amino acid modification comprises modification of an amino acid residue selected from an aspartic acid residue, a glutamic acid residue, 21 ine residue, a serine residue, a threonine residue, an asparagine residue, a glutamine residue, a cysteine residue, or a tyrosine residue (as described above), the ano acid residue is substituted with a lysine residue or an arginine residue. Thus, in [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar one embodiment, a negatively charged residue or a polar e is tuted with a positively charged residue, thus increasing the ratio of positive to negative charges and increasing the pI of the clostridial toxin.
In one embodiment, the at least one amino acid modification (as described above) comprises modification of an asparagine amino acid residue or a glutamine amino acid residue (both uncharged, polar residues). In one embodiment, the gine or glutamine amino acid residue is tuted with a lysine residue or an arginine residue (both positively charged residues). In one embodiment, the asparagine or ine amino acid residue is substituted with a lysine e. In one embodiment, the asparagine or glutamine amino acid e is substituted with an arginine residue. gine and glutamine residues are advantageously suitable for modification as they are polar, form only weak dipole interactions with other residues, and constitute 14% of a l clostridial toxin molecule (such as BoNT/A).
In one embodiment, the engineered clostridial toxin is a BoNT/A. A nce BoNT/A sequence has the UniProtKB ion Number P10845.
The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/A clostridial toxin.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20 or 25) amino ) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, and GLN 1229, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an ise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an amine residue. In one embodiment, said modification comprises substitution of the [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar amino acid with a lysine e. In one embodiment, said modification comprises substitution of the amino acid with an arginine e.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, and GLN 1229, and said amino acid modification(s) increase(s) the ctric point (pl) of the engineered BoNT/A to a value that is at least 0.5 (for e, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an ise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one ment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, and GLN 1229, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine e or an arginine residue. In one embodiment, said modification ses tution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a , said eQeered BoNT/A comprises a modification of at least one (for example, at least 1, [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar 2, 3, 4, 5, 10, 15, or 20) amino acid(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, and ASP 1086; and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises tution of the amino acid with a lysine e. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, n the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a ation of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30) amino acid(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, and said amino acid modification(s) increase(s) the ctric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one ment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one ment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, n the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30) amino acid(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, AD 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, and said amino acid modification(s) increase(s) the isoelectric point (p1) of the engineered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine e. In one ment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, n the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30) amino acid(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the ered BoNT/A to a value that is at least 1 p1 unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine e. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification ses substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a , said ered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino acid(s) selected from: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one endiment said modification comprises substitution of the amino acid with a lysine [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar ation] r MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue, In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one ment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino acid(s) selected from: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an ise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an ne residue. In one embodiment, said modification ses substitution of the amino acid with a lysine residue. In one embodiment, said modification ses substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino acid(s) selected from: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and said amino acid modification(s) increase(s) the ctric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s), In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises tution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of the following seven amino acids: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0Q9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by r [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar said amino acid modifications. In one embodiment said modification ses substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises tution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the ered idial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of the following seven amino acids: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications. In one embodiment said modification ses substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one ment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said ered BoNT/A comprises a ation of the ing seven amino acids: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications. In one embodiment said modification comprises tution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino acid(s) selected from: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modification(s) increase(s) tnoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an ise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one ment, said modification ses substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the ered clostridial toxin is a BoNT/A, said engineered BoNT/A ses a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino acid(s) selected from: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said ation comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a , said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino acid(s) selected from: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises tution of the amino acid with a lysine residue or an ne residue. In one embodiment, said modification comprises tution of the amino acid with a lysine residue, In one embodiment, said ation comprises substitution of the amino acid with an 3O arginine residue.
In one embodiment, wherein the engineered idial toxin is a BoNT/A, said Afi 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229; anden ineered BoNT/A comprises a modification of the following seven amino acids: [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications. In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one ment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a , said engineered BoNT/A ses a modification of the following seven amino acids: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modifications se the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications. In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, n the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of the following seven amino acids: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modifications increase the ctric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications. In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one ment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered idial toxin is a BoNT/A, said d BoNT/A comprises a modification of at least one (for example, at least 1, [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r 2, 3, 4, 5, or 6) amino acid(s) ed from: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0,4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s).
In one embodiment said modification comprises substitution ofthe amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one ment, wherein the engineered clostridial toxin is a , said engineered BoNT/A ses a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino acid(s) selected from: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid ation(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said ation comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a , said ered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, or 6) amino ) selected from: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of the following seven amino acids: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229; and said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications, In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises tution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of the following seven amino acids: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modifications increase the ctric point (pI) of the ered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid ations. In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said ation ses substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one ment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of the following seven amino acids: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, or GLN 1229, and said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications. In one embodiment said modification comprises tution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification ses snitution of the amino acid with an ne residue.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar In one embodiment, wherein the engineered idial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 6 or 7) amino acid(s) selected from: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the ered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an ne residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said ered BoNT/A comprises a ation of at least one (for example, at least 1, 2, 3, 4, 5, 6 or 7) amino acid(s) selected from: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an ise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an ne residue. In one embodiment, said modification comprises tution of the amino acid with a lysine residue. In one ment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a , said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 6 or 7) amino acid(s) selected from: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification curises substitution of the amino acid with a lysine residue or an arginine residue.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar In one embodiment, said modification comprises substitution of the amino acid with a lysine e. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of the ing eight amino acids: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277, and said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A lacking said amino acid modifications. In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue.
In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said ered BoNT/A comprises a modification of the following eight amino acids: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277; and said amino acid modifications increase the isoelectric point (pI) of the engineered BoNT/A to a value that is at least 0.5 (for e, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/A g said amino acid modifications. In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one ment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said ation ses substitution of the amino acid with an arginine residue.
In one embodiment, wherein the ered clostridial toxin is a , said engineered BoNT/A comprises a modification of the ing eight amino acids: ASN 1188, ASP 1213, GLY 1215, ASN 1216, ASN 1242, ASN 1243, SER 1274, THR 1277, and said amino acid modifications increase the isoelectric point (pI) of the (efiir’l‘ejred BoNT/A to a value that is at least one pI unit higher than the pI of anise identical BoNT/A lacking said amino acid modifications. In one [Annotation] wilksar None set by wilksar ation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar embodiment said ation ses substitution of the amino acid with a lysine residue or an arginine e. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one ment, said modification ses substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 6, or all 7) amino acid(s) selected from: ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, HIS 1064, and GLN 1229, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue.
In one embodiment said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment said modification comprises tution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11) amino acid(s) selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, HIS 1064, ASN 1080, ASN 1147, and GLN 1229; and said amino acid modification(s) increase(s) the isoelectric point (p1) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine e or an arginine residue. In one ment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification ses substitution of the amino acid with an ne residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13) amino acid(s) selected from: ASN 886, ASN 905 ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, In 1064, ASN 1080, ASN 1147, and GLN 1229; and said amino acid [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/A to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine e or an arginine residue.
In one embodiment said ation comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine e.
In one embodiment, the engineered clostridial toxin is a BoNT/B. A reference BoNT/B sequence has the UniProtKB Accession Number P10844.
The present inventors have fied certain amino acids that represent preferred targets for amino acid modification in a BoNT/B clostridial toxin.
In one embodiment, wherein the engineered idial toxin is a BoNT/B, said engineered BoNT/B ses a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252, and ASN 1273, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/B to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/B lacking said amino acid modification(s). In one ment said ation comprises substitution of the amino acid with a lysine residue or an arginine e. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered idial toxin is a BoNT/B, said engineered BoNT/B comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, [in 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] wilksar ed set by wilksar 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252, and ASN 1273; and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/B to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/B lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one ment said modification comprises substitution of the amino acid with an ne residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/B, said engineered BoNT/B comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252, and ASN 1273, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/B to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/B g said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one ment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, the engineered clostridial toxin is a BoNT/Cl. A reference BoNT/C1 ce has the UniProtKB Accession Number .
The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/C1 clostridial toxin.
Thus, in one ment, wherein the engineered clostridial toxin is a BoNT/Cl, said engineered BoNT/C1 ses a modification of at least one (for example, at least 1, 2 3 4, 5, 10, or 15) amino acid selected from: ASN 881, ASP 898, GLU 916, GLU ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081; and said amino acid ation(s) increase(s) the isoelectric point (pI) of the engineered BoNT/C1 to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of an otherwise identical BoNT/C1 g said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine e. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine e.
Thus, in one embodiment, wherein the engineered clostridial toxin is a BoNT/Cl, said engineered BoNT/C1 ses a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/C1 to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/C1 lacking said amino acid ation(s). In one embodiment said modification comprises tution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/Cl, said engineered BoNT/C1 comprises a ation of at least one (for e, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081, and said amino acid ation(s) increase(s) the isoelectric point (pI) of the engineered BoNT/C1 to a value that is at least one pI unit higher than the pI of an otherwise identical 1 lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an alpine residue. In one embodiment, said modification comprises substitution of the [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] r None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar ed set by wilksar amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, the engineered clostridial toxin is a BoNT/D. A reference BoNT/D sequence has the UniProtKB Accession Number P19321.
The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/D clostridial toxin.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/D, said engineered BoNT/D ses a modification of at least one (for e, at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122; and said amino acid modification(s) increase(s) the ctric point (pI) of the engineered BoNT/D to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 017, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/D lacking said amino acid modification(s). In one ment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine e. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/D, said engineered BoNT/D comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/D to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/D lacking said amino acid modification(s). In one embodiment said modification comprises tution of the amino acid with a lysine residue or an amine residue. In one embodiment, said modification comprises substitution of the [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar amino acid with a lysine e. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, n the engineered clostridial toxin is a BoNT/D, said engineered BoNT/D comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122; and said amino acid modification(s) increase(s) the ctric point (pI) of the engineered BoNT/D to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/D lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine e or an ne residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, the engineered clostridial toxin is a BoNT/E. A reference BoNT/E sequence has the UniProtKB Accession Number Q00496.
The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/E clostridial toxin.
In one embodiment, wherein the engineered idial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/E lacking said amino acid modification(s). In one ment said modification ses snitution of the amino acid with a lysine e or an arginine residue. In one ation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar embodiment, said modification comprises substitution of the amino acid with a lysine e. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/E to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said ation comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine e. In one embodiment said modification ses substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, or 25) amino acid selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140, and said amino acid modification(s) se(s) the isoelectric point (pI) of the engineered BoNT/E to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an 3O arginine residue. In one embodiment, said ation comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
[Annotation] r None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar In one embodiment, the engineered clostridial toxin is a BoNT/F. A reference BoNT/F sequence has the UniProtKB Accession Number YP_001390123.
The present inventors have identified certain amino acids that ent preferred s for amino acid modification in a BoNT/F idial toxin.
In one embodiment, wherein the ered clostridial toxin is a BoNT/F, said engineered BoNT/F comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/F to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/F g said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/F, said ered BoNT/F comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/F to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 unit higher than the pI of an otherwise identical BoNT/F lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue.
In one ment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said ation comprises substitution of the amino acid with an arginine residue. ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar In one embodiment, wherein the engineered clostridial toxin is a BoNT/F, said engineered BoNT/F comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, or 20) amino acid selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056, and said amino acid modification(s) increase(s) the ctric point (pI) of the engineered BoNT/F to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/F g said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, the engineered clostridial toxin is a BoNT/G. A reference BoNT/G sequence has the UniProtKB Accession Number Q60393.
The present inventors have identified certain amino acids that represent preferred targets for amino acid ation in a BoNT/G clostridial toxin.
In one embodiment, wherein the engineered clostridial toxin is a BoNT/G, said engineered BoNT/G comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 1090, and said amino acid ation(s) increase(s) the isoelectric point (pI) of the engineered BoNT/G to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/G lacking said amino acid modification(s). In one embodiment said modification ses substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine e. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
[Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar In one ment, wherein the engineered clostridial toxin is a , said engineered BoNT/G ses a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 1090, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/G to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 units higher than the pI of an otherwise identical BoNT/G lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one embodiment, wherein the ered clostridial toxin is a BoNT/G, said ered BoNT/G ses a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 1090, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered BoNT/G to a value that is at least one pI unit higher than the pI of an otherwise identical BoNT/G lacking said amino acid modification(s). In one embodiment said modification comprises tution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one ment, the engineered clostridial toxin is a TeNT. A reference TeNT sequence has the UniProtKB Accession Number P04958.
The present inventors have identified certain amino acids that represent preferred targets for amino acid ation in a TeNT clostridial toxin.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] r Unmarked set by wilksar In one embodiment, n the engineered clostridial toxin is a TeNT, said engineered TeNT comprises a ation of at least one (for example, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered TeNT to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 unit higher than the pI of an otherwise identical TeNT lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said ation comprises substitution of the amino acid with an arginine e.
In one embodiment, wherein the engineered clostridial toxin is a TeNT, said ered TeNT comprises a modification of at least one (for e, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097; and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered TeNT to a value that is at least 0.5 (for example, at least 0.5, 0.6, 0.7, 0.8, 0.9 or 1) p1 unit higher than the pI of an otherwise identical TeNT lacking said amino acid ation(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment said modification comprises substitution of the amino acid with an arginine residue.
In one ment, wherein the engineered clostridial toxin is a TeNT, said engineered TeNT ses a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, or 15) amino acid selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097, and said amino acid modification(s) increase(s) the isoelectric point (pI) of the engineered TeNT to a value tns at least one pI unit higher than the pI of an otherwise identical TeNT lacking [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar ation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said ation comprises substitution of the amino acid with a lysine residue. In one ment said modification comprises substitution of the amino acid with an arginine residue.
The present invention is suitable for application to many different varieties of clostridial toxin. Thus, in the context of the present invention, the term "clostridial toxin" embraces toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F and G), C. lelani (tetanus neurotoxin), C. bulyricum (botulinum neurotoxin serotype E), and C. baratil' (botulinum neurotoxin serotype F), as well as modified clostridial toxins or derivatives derived from any of the foregoing. The term "clostridial toxin" also embraces botulinum neurotoxin serotype H. num neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex, consisting of BoNT itself complexed to a number of accessory proteins. There are at present eight different classes of botulinum neurotoxin, namely: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and H, all of which share similar structures and modes of . Different BoNT serotypes can be distinguished based on inactivation by specific neutralising anti-sera, with such classification by serotype ating with percentage sequence identity at the amino acid level. BoNT ns of a given serotype are further divided into different subtypes on the basis of amino acid percentage sequence identity.
BoNTs are absorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and t the release of their neurotransmitter acetylcholine. BoNT/B, , BoNT/F and BoNT/G cleave obrevin/vesicle-associated ne n (VAMP), 1, BoNT/A and BoNT/E cleave the synaptosomal-associated protein of 25 kDa (SNAP—25); and BoNT/C1 cleaves syntaxin.
Tetanus toxin is produced in a single serotype by C. tetam’. C. butyricum produces BoNT/E, while C. baratil' produces BoNT/F.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar The term "clostridial toxin" is also intended to embrace 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 has been modified as compared to the native ified) form of the clostridial toxin, or may contain one or more inserted amino acids that are not present in the native ified) form of the clostridial toxin. By way of example, a d clostridial toxin may have modified amino acid sequences in one or more s relative to the native (unmodified) clostridial toxin sequence. Such modifications may modify functional aspects of the toxin, for e biological ty or tence. Thus, in one embodiment, the engineered clostridial toxin of the invention is an engineered modified clostridial toxin, or an engineered modified clostridial toxin derivative, or an engineered clostridial toxin derivative.
A modified clostridial toxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a d Hc domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial toxin. Such modifications in the Hc domain can include modifying residues in the ganglioside binding site of the HC domain or in the protein (SV2 or synaptotagmin) binding site that alter binding to the ganglioside receptor and/or the n receptor of the target nerve cell. Examples of such modified clostridial toxins are bed in and WC 2006/114308, both of which are hereby incorporated by nce in their entirety.
A modified idial toxin may have one or more modifications in the amino acid sequence of the light chain, for example modifications in the substrate binding or catalytic domain which may alter or modify the SNARE protein specificity of the modified LC. Examples of such d clostridial toxins are described in WO 2010/120766 and US 318385, both of which are hereby incorporated by reference in their entirety.
A modified clostridial toxin may comprise one or more modifications that increases or decreases the biological activity and/or the biological persistence of the modified clostridial toxin. For example, a modified clostridial toxin may comprise a leucine- or tDine-based motif, wherein said motif increases or decreases the ical activity [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] r None set by r [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar and/or the biological persistence of the modified clostridial toxin. le leucine— based motifs include xDxxxLL, L, xExxxIL, and xExxxLM (wherein x is any amino acid). Suitable tyrosine-based motifs include Y-x-x-Hy (wherein Hy is a hydrophobic amino acid). Examples of modified idial toxins comprising leucine- and tyrosine—based motifs are described in W0 2002/08268, which is hereby incorporated by reference in its entirety.
The term "clostridial toxin" is intended to embrace hybrid and chimeric clostridial toxins. A hybrid clostridial toxin comprises at least a portion of a light chain from one clostridial toxin or subtype thereof, and at least a portion of a heavy chain from another clostridial toxin or clostridial toxin subtype. In one embodiment the hybrid clostridial toxin may contain the entire light chain of a light chain from one clostridial toxin subtype and the heavy chain from another idial toxin subtype. In another embodiment, a chimeric clostridial toxin may contain a portion (e.g. the binding domain) of the heavy chain of one clostridial toxin e, with another portion of the heavy chain being from another clostridial toxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain ns from different clostridial toxins. Such hybrid or chimeric clostridial toxins are useful, for example, as a means of delivering the therapeutic s of such idial toxins to patients who are immunologically resistant to a given clostridial toxin subtype, to patients who may have a lower than average concentration of receptors to a given clostridial toxin heavy chain binding domain, or to patients who may have a protease-resistant variant of the membrane or vesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin).
Hybrid and chimeric clostridial toxins are described in US 8,071,110, which publication is hereby incorporated by reference in its entirety. Thus, in one embodiment, the engineered clostridial toxin of the invention is an ered hybrid clostridial toxin, or an engineered chimeric idial toxin.
The term "clostridial toxin" is intended to embrace re-targeted clostridial toxins. In a 3O re-targeted clostridial toxin, the clostridial toxin is modified to include an ous ligand known as a Targeting Moiety (TM). The TM is selected to provide binding specificity for a desired target cell, and as part of the re-targeting process the native binding portion of the clostridial toxin (e.g. the He domain, or the Hcc domain) may bnmoved. Re—targeting technology is described, for example, in: EP-B—O689459; [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar W0 1994/021300, EP-B-O939818; US 6,461,617, US 7,192,596; W0 1998/007864; EP-B-O826051, US 5,989,545, US 6,395,513; US 6,962,703, , EP- B-O996468; US 7,052,702, W0 1999/017806; EP-B-1107794; US 6,632,440; W0 2000/010598, WO 1213, WO 2006/059093, W0 2000/62814, W0 2000/04926; ; ; and ; all of which are hereby incorporated by reference in their entirety. Thus, in one embodiment, the engineered clostridial toxin of the invention is an engineered re-targeted clostridial toxin.
The present ion also embraces clostridial toxins that have a non-native protease cleavage site. In such idial toxins, the native protease cleavage site (also known as the activation site, as described above) is d or replaced With a protease cleavage site that is not native to that clostridial toxin (i.e. an exogenous cleavage site). Such a site Will require an exogenous protease for cleavage, Which allows for improved control over the timing and location of ge events. Non-native protease cleavage sites that may be employed in clostridial toxins include: kinase (DDDDKi) Factor Xa (IEGRi / IDGRi) TEV(Tobacco Etch virus) (ENLYFQLG) Thrombin (LVPRiGS) ssion (LEVLFQLGP).
Additional protease cleavage sites include recognition sequences that are cleaved by a non-cytotoxic protease, for example by the light chain of a clostridial neurotoxin.
These include the SNARE (eg. 5, syntaxin, VAMP) protein recognition sequences that are d by non-cytotoxic proteases such as the light chain of a clostridial neurotoxin. Clostridial toxins sing non-native 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. Also ed by the term se cleavage site is an intein, which is a self-cleaving sequence. The self- splicing reaction is controllable, for example by varying the concentration of reducing agent present.
The present invention also embraces clostridial toxins comprising a "destructive cnage site". In said clostridial toxins, a non-native protease cleavage site is [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar ation] wilksar None set by wilksar ation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar incorporated into the clostridial toxin, at a location chosen such that cleavage at said site will decrease the activity of, or vate, the clostridial toxin. The destructive protease cleavage site can be susceptible to cleavage by a local protease, in the event that the clostridial toxin, following administration, es to a non-target location.
Suitable non-native protease cleavage sites include those described above. Clostridial toxins comprising a destructive cleavage site are described in and , both of which are hereby incorporated by reference in their entirety.
The engineered clostridial toxins of the present invention, especially the light chain component thereof, may be PEGylated — this may help to increase stability, for example duration of action of the light chain component. PEGylation is particularly preferred when the light chain comprises a BoNT/A, B or C1 protease. PEGylation preferably includes the addition of PEG to the N—terminus of the light chain component. By way of example, the inus of a light chain may be extended with one or more amino acid (e. g. cysteine) es, which may be the same or different.
One or more of said amino acid residues may have its own PEG molecule attached (e.g. covalently attached) thereto. An example of this technology is described in W02007/104567, which is hereby incorporated by reference in its entirety.
The ered clostridial toxins of the present invention may be free from the xing proteins that are present in a naturally occurring clostridial toxin complex.
An engineered clostridial toxin of the present invention may also comprise a limited number of non-standard amino acids. Thus, in on to the 20 standard amino acids, non-standard amino acids (such as oxyproline, 6-N-methyl lysine, 2- aminoisobutyric acid, isovaline and u—methyl serine) may be substituted for amino acid residues of the engineered clostridial toxins of the present invention. A limited 3O number of non—conservative amino acids, amino acids that are not encoded by the genetic code, and ral amino acids may be substituted for clostridial polypeptide amino acid residues. The ered clostridial toxins of the present invention can also comprise non-naturally occurring amino acid residues.
[Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Non-naturally occurring amino acids e, t limitation, trans—3- methylproline, 2,4-methano-proline, cishydroxyproline, transhydroxy-proline, N-methylglycine, allo-threonine, -threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert- 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, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and ation of ds containing nonsense ons is d out in a cell free system comprising an E. coli S30 t and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. $2722, 1991, Ellman et al., Methods l. @301, 1991; Chung et al., Science @1806—9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 45-9, 1993). In a second method, translation is carried out in s oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. fl:19991-8, 1996). Within a third method, E. coli cells are cultured in the e of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3—azaphenylalanine, 4—azaphenylalanine, or 4- fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. fiz7470-6, 1994.
The engineered clostridial toxins of the present invention can be produced using recombinant nucleic acid technologies. Thus, in one embodiment, an engineered clostridial toxin (as described above) is a recombinant engineered clostridial toxin. 3O In another aspect, the present invention provides a nucleic acid (for example, a DNA) comprising a nucleic acid sequence encoding an engineered clostridial toxin as described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator.
[Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar In a preferred embodiment, the vector has a promoter selected from: Promoter ion Agent Typical Induction ion Tac (hybrid) IPTG 0.2 mM 2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0mM) In another preferred embodiment, the vector has a promoter selected from: Promoter Induction Agent Typical Induction ion Tac (hybrid) IPTG 0.2 mM (0.05-2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0mM) T5-lac operator IPTG 0.2 mM (0.05-2.0mM) The nucleic acid molecules of the invention may be made using any suitable process known in the art. Thus, the nucleic acid molecules may be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention may be made using molecular biology techniques.
The DNA construct of the present invention is ably ed in silica, and then synthesised by conventional DNA synthesis techniques.
The above-mentioned nucleic acid sequence information is optionally modified for codon—biasing according to the ultimate host cell (e. g. E. coli) expression system that is to be employed.
In one embodiment, the c acid sequence encoding an engineered clostridial toxin as described above is a nucleic acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98 or 99%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7 and 9.
In one aspect, the present invention es a nucleic acid sequence having at least 7D(for example, at least 75, 80, 85, 90, 95, 97, 98 or 99%) sequence identity to a [Annotation] wilksar None set by wilksar [Annotation] r MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by r nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7 and 9. In one embodiment, the nucleic acid sequence has at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7 and 9.
The present invention also provides polypeptides encoded by c acid ces as described above. Thus, in one aspect, the present invention provides a polypeptide sing an amino acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98 or 99%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 6, 8 and 10. In one embodiment, the amino acid sequence has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 6, 8 and 10.
In one embodiment, the engineered clostridial toxin of the invention is an engineered BoNT/A as bed above, and said engineered BoNT/A comprises (or consists of) an amino acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequence identity to an amino acid sequence ed from SEQ ID NOs: 4, 6, 8 and 10.
In one embodiment, the engineered clostridial toxin of the ion is an engineered BoNT/A as described above, and said engineered BoNT/A comprises (or consists of) the amino acid sequence of SEQ ID NO: 4, 6, 8 or 10.
In one aspect, the invention provides a polypeptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 4, 6, 8 or 10, In one aspect, the invention provides a c acid ng an engineered clostridial toxin as described above, wherein said nucleic acid comprises a nucleic acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequence ty to a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 3O 7 and 9. In one embodiment, the nucleic acid comprises (or consists of) the nucleic acid sequence of SEQ ID NO: 3, 5, 7 or 9.
In one aspect, the invention provides a nucleic acid comprising (or consisting of) the nuic acid sequence of SEQ ID NO: 3, 5, 7 or 9.
[Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar The "percent sequence identity" n two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the ces.
Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. ations of % sequence identity may also take into t the number of gaps, and the length of each gap that needs to be introduced to optimize ent of two or more sequences. Sequence isons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.
In one aspect, the present ion es a method of producing a single-chain engineered clostridial toxin protein having a light chain and a heavy chain, the method comprising expressing a nucleic acid (said c acid being as described above) in a suitable host cell, lysing the host cell to provide a host cell homogenate containing the single-chain engineered clostridial toxin protein, and isolating the —chain engineered clostridial toxin protein.
In another aspect, the present invention provides a method of activating an engineered clostridial toxin, the method comprising providing a single-chain engineered clostridial toxin protein obtainable by the method of producing a single-chain engineered clostridial toxin protein as bed above, contacting the polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the light chain and heavy chain, thereby converting the polypeptide into a di—chain polypeptide wherein the light chain and heavy chain are joined together by a disulphide bond.
The engineered clostridial toxins of the invention may be used to prevent or treat certain l or cosmetic diseases and conditions. Thus, in a further aspect, the present invention provides an engineered clostridial toxin as described above, for use in medicine.
In a related aspect, the present invention provides an engineered clostridial toxin as described above, for use in the prevention or treatment of a disease or condition sated from: strabismus, blepharospasm, squint, ia (e.g. spasmodic dystonia, [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal ia, limb dystonia, cervical dystonia), ollis (e.g. spasmodic torticollis), beauty therapy (cosmetic) applications benefiting from uscle incapacitation (via SNARE down- regulation or inactivation), neuromuscular disorder or condition of ocular motility (e.g. concomitant strabismus, vertical strabismus, l rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tics, tal myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, charley horse, n headaches, r pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's e, stuttering, hemifacial spasm, eyelid disorder, al palsy, focal city, dic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, ive salivation, excessive intestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e. g. tension headache), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological ers, chronic neurogenic inflammation, and a smooth muscle disorder.
In use, the present invention employs a pharmaceutical composition, comprising an engineered clostridial toxin, together with at least one component selected from a pharrnaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.
The engineered clostridial toxins of the present invention may be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.
In the case of an engineered clostridial toxin that is to be delivered locally, the ered clostridial toxin may be ated as a cream (e.g. for topical application), or for sub-dermal injection.
Local delivery means may include an aerosol, or other spray (e.g. a nebuliser). In this regard, an aerosol formulation of an engineered idial toxin enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] r ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Engineered clostridial toxins of the invention may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of an affected organ.
A preferred route of administration is via laproscopic and/ or localised, particularly intramuscular, injection.
The dosage ranges for administration of the engineered clostridial toxins of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the engineered clostridial toxin or ition, the route of stration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient’s ion, contraindications, if any, and the judgement of the attending physician. ions in these dosage levels can be adjusted using rd empirical routines for optimisation.
Suitable daily dosages (per kg weight of patient) are in the range 0.0001-1 ng/kg, preferably -0.5 ng/kg, more preferably 0.002-05 ng/kg, and particularly preferably 0.004-0.5 ng/kg. The unit dosage can vary from less than 1 picogram to 30ng, but typically will be in the region of 0.01 to 1 ng per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly.
A particularly preferred dosing regimen is based on 0.05 ng of engineered clostridial toxin as the 1X dose. In this regard, preferred dosages are in the range lX—lOOX (i.e. 0.05-5 ng).
Fluid dosage forms are typically ed utilising the engineered clostridial toxin and a pyrogen-free sterile vehicle. The engineered clostridial toxin, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. In preparing solutions the ered clostridial toxin can be dissolved in the vehicle, the on being made ic if necessary by addition of sodium chloride and sterilised by filtration h a e filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is :damate, the solution in its sealed containers may be sterilised by aving. ntageously additives such as buffering, solubilising, stabilising, preservative or ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r ed set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar ation] wilksar Unmarked set by wilksar bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.
Dry powders, which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre—sterilised ingredients into a sterile container using aseptic technique in a sterile area, Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.
Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in ntially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and isation cannot be accomplished by filtration. The components may be isolated in a sterile state or atively it may be ised after isolation, e.g. by gamma ation.
Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition(s) to facilitate uniform distribution of the components.
Administration in accordance with the present invention may take advantage of a variety of delivery technologies including microparticle encapsulation, viral delivery systems or high-pressure aerosol impingement.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by r [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar List of Figures Figure 1 Isoelectric focusing (IEF) gel of cationic ucts.
Figure 2 Percentage SNAP-25 cleavage in rat embryonic spinal cord s (eSCN) for Cat5v2(K1064H/N954K) (A), (K1064H/N886K) (B) and Cat5v2(K1064H/ N1025K) (C), and summary of pECSO relative to nBoNT/Al. (A, B, C) Rat embryonic spinal cord neurons were cultured for three weeks and treated with Cat5v4 for 24 h, before Western blotting with SNAP-25 specific antibody. Data is mean ::SEM from three independent experiments in triplicate. (D) Relative potency of Cat5v2(K1064H/N886K), Cat5v2(K1064H/N954K) and Cat5v2(K1064H/ N1025K) to Al (List ical Laboratories) in the rat eSCN SNAP-25 cleavage potency assay. Each point corresponds to an individual batch and is a mean of 3 independent pECSO determinations based on an 8-point concentration response curve (CRC). Each concentration in the CRC was assessed in triplicate. Potency comparisons are made to a mean of List batches, pooled data n=24. Data are mean iSEM of n=3 batches per Cat5v4.
Figure 3 The potency (tso) of nBoNT/Al and Cat5v4 in the mouse phrenic nerve hemi- diaphragm assay (mPNHD). Mouse phrenic nerve hemi-diaphragm tissue was ted with Cat5v4 or native BoNT/Al as indicated. Diaphragm contractile force was ed until the contraction was no longer detectable or after 140 minutes.
Each point corresponds to independent determinations. The tso value is the time required to inhibit the contractile force of the mouse hemi-diaphragm by 50%.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Seguences SEQ ID NO: 1. BoNT/Al nucleic acid sequence.
ATGCCATTCGTCAACAAGCAA"1TCAACTACAAAGACCCAGTCAACGGCGTCGACA"1CGCATACATCAAG ATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCG GAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCT "1ACTACGAT GTACCTGAGCACGGATAACGAAAAAGATAACTACC"1GAAAGGTGTGACC AAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCA‘L-1T GTTCGCGGTATC CCGTTCTGGGGTGGTAGCACGAT"1 GACACCGAAC"1GAAGGTTATCGACACTAACTGCATTAACGTTATT CAACCGGATGGTAGC"1ATCGTAGCGAAGAGCTGAATCTGGTCATCAT"1 GGCCCGAGCGCAGACATTATC CAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAG TACAT"1CGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTG C'-'GGG‘_' GCGGGCAAA"_1TCGCTACCGATCCGGC"-1 GT CACGCTGGCCCATGAACTGATCCACGCAGGCCAC CGCCTGTACGGCA"1TGCCATCAACCCAAACCGTGTGTTCAAGGTTAA"1ACGAA"1 GCATACTACGAGATG AGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCA"1 GACGCTAAATTCATTGACAGC TTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAG GCCAAAAGCATCGTTGGTACTACCGCGTCGT"1 GCAGTATATGAAGAA"1 GTGTTTAAAGAGAAGTACCTG CTGTCCGAGGATACCTCCGGCAAGTT"_1AGCGT-"1GATAAGCTGAAGTT-"1GACAAACTGTACAAG "1G-CT G ACCGAGATTTACACCGAGGACAACTT"1 GTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAAT TTTGACAAAGCGGTT"1T CAAGATTAACATCGTGCCGAAGGTGAACTACACCA"1CTATGACGGTTTTAAC CTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACG AAGTTGAAGAACTTCACGGGTCTGTTCGAGTTC"1ATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGC AAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAAT_TGG GATC' ‘GTT C'i‘T T' ‘CGCCAT CCGAAGATAA".1 T TTACCAACGACCTGAACAAGGG".1 GAAGAAA"-1CACCAGC GATACGAATA"1 T GAAGCAGCGGAAGAGAA"1A"1CAGCC"1 GGATCTGATCCAGCAGTACTATCTGACC‘ 'T' .
AACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAA CCGAATA"1 CGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCAT CGTGCACAGGAGTT "CGAACACGG‘L1AAAAGCCGTATCGCGC'L1 GACCAACAGCGTTAACGAGGCC AACCCGAGCCG'-'GT C‘-'A"1ACCTTCT"1 CAGCAGCGACTATGTTAAGAAAG"1 GAACAAAGCCACT GAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTG AGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGC AACATGCTGTACAAAGACGATT "1TGTGGG"1GCCCTGATCTTCTCCGGTGCCGTGA"1T CTGCTGGAGTTC ATTCCGGAG TTGCGATCCCGGTGTTGGG"1ACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTG .1CAGACCA‘-' CGA'JAACGCGC".1GTCGAAACGTAATGAAAAA‘TGGGACGAGG"--1TTACAAATACATT AATTGGCTGGCGAAAGTCAA"1ACCCAGA"1CGACCTGATCCGTAAGAAAA"1GAAAGAGGCGCTG GAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAG AATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATC AATATCAACAAGTTTT"1GAATCAGT G"1AGCGTTTCGTACCTGATGAA"1AGCATGA"1TCCGTATGGCGTC AAACG"-1 CTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAA"-1CGTGGT 40 ACGCTGATTGGCCAAGT-"1GACCGCT-"1GAAAGACAAAGTTAACAATACCC"-1 GAGCACCGACAT CCCATT "1 CAACTGAGCAAGTATGTTGATAATCAACGTCTGT"1GAGCACTTTCACCGAGTATA"1 CAAAAACATCATC AATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAG ATCAACATCGGTAGCAAGGTCAAT"1TTGACCCGATCGATAAGAACCAGATCCAGC"1GTTTAATCTGGAA TCGAGCAAAA'L1 T GAGGTTAT CCTGAAAAACGCCATTGTCTACAACT CCATGTACGAGAAT-T"1-CTCCACC 45 AGCTTCTGGATTCGCATCCCGAAA"-1ACT"1CAACAGCA".1 TAGCCTGAACAACGAGTATACTATCATCAAC TGTATGGAGAACAACAGCGG"1TGGAAGG"1GT CT C' 'GAAC' ‘ATGGTGAGATCATTTGGACCTTGCAGGAC ACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAAT CGTTGGATCTTCGTGACCATTACGAATAACCG"1 cr'l CAGCAAGATTTACA"1CAATGGTCGCTTG ATCGATCAGAAACCGATTAGCAACCTGGGTAA"-1AT CCACGCAAGCAACAACATTA"-1GTTCAAATTGGAC 50 CGCGATACCCATCG' "-'ATATCTGGATCAAGTATTTCAACCTGT-"1TGATAAAGAACTGAATGAG AAGGAGATCAAAGAT' ‘TG‘i'A' CCAATCTAACAGCGGCAT"1TTGAAGGACT "1CTGGGGCGATTAT CTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAAT GTGGG"1AT"1 CGT GGTTACATGTATTTGAAGGG"1 CCGCGTGGCAGCGTTATGACGACCAACAT "1TACCTG AACTC"-1AGCCTGTACCGTGGTACGAAAT-TCATCATTAAGAAATATGCCAGCGGCAACAAAG TAACATT 55 G'-'GCG‘-AA-AACGATCG-'GT C‘-'ACA'-'CAACG"1 GGT CGT GAAGAA"-1AAAGAGTACCGTCTGGCGACCAAC GCTTCGCAGGCGGGTGTTGAGAAAAT' 'C' ‘GAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAA GTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAAC Ans"AACGACATCGGCTTTATTGG"1TTCCACCAGT"1CAACAA"1ATr1Gcr1AAACTGGTAGCGAGCAAT [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar TGGTACAATCGTCAGAT"1GAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGAT GATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 2. BoNT/Al amino acid sequence.
QFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGJLNPPPEAKQV_D V DNYLKGVTKL STD.JGRVILL"1SIVRGI PFWGGSTIDTELKVIDTNCINVI Q?DGSYRSEELNLVIIGPSA DI IQFECKS FGHEVLNLTRNGYGSTQYIRTSPDFTFGTEESLEVDTNRJ LGAGKFATD?AVTLAHELIHAGHRLYGIAINPNQVFKVNTNAYYEMSGLEVSFEE.JRTFGGHDAKTI DS .YYYNKF < DIAST KSIVGTTAS .LQYWKNVFKEKYL.LSE DTSGKFSV DKLKFDKLYKM__| .‘ _l I YTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNY'L1IY3GJ:N_.RN'l'NLAANbNGQNTEINNMNb-T LFEFY GI ITSKT {SLDKGYN {ALNDLCIKVNNWLJLFFSPSEDNFTND.JNKGE.LITS TAAEEVIS .D'IIQQYY )NEPENISIEN.JSSDI IGQLE.4MP\IIERFPNG (KYELDKYTMF-I LRAQEFEHGKSQIALTNSVNEALLN?SRVYTFTSSDYVKKVNKATEAAMTLGWVEQLVYDFTDETSEV STT 3 {IA PYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKV._l HVQTIDNALSKRNEKWDEVYKYIVL1NWLAKVNTQL D_.I RKKMKEALENQAEATKAI INYQYNQYTEEEK I DDLSS {LNESINKAMINLNKFLNQCSVSYLMNSMI PYGVKRLEDFDAS LK DAL.JKYIYJNRG H .JIGQVD QLKDKVNVTLSTDI?FQLS {YVDNQRLLSTFTEYI {NI INTS I .N .RYESNHLIDLSRYAS < H NFDPIDKVQIQLFNLESSKIEVIT.KNAIVYNSMYEWFSTSFWI?I?KYFNSISLNNEYTIIN OMEVNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINIS DYINRWI FVT ITNNR.LNNSKIYINGR._.
HDQKPISNLGNIflASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGLLKDFWGDYQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFILKKYASGNKDNLQNNDRVYINVVVKVKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVM{SKNDQGITNKCKMNJQDNGNDIGFIGFHQTNVIAKLVASNWYVQQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO: 3. ered BoVT/Al "Cat—A" nucleic acid sequence.
A-1GCCA"-1 T C GT CAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACA'L1 CGCATACATCAAG ATTCCGAACGCCGG".1CAAATGCAGCCGGTTAAGGCTTr1 TAAGATCCACAACAAGAT-TTGGGTTATCCCG GAGCGTGACACCTTCACGAACCCGGAAGAAGGCGAT Cr1GAACCCGCCACCGGAAGCGAAGCAAGTCCC"1 GTCAGCTACTACGAT r1 CGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACC GTTCGAACG’1ATCTACAGCACGGATCTGGGTCGCAr1 GCT GCTGACTAGCATTGTTCGCGGTATC CCGT"1CTGGGGTGG"1AGCACGATTGACACCGAAC"1GAAGGr1TATCGACACTAACTGCATTAACGTTATT CAACCGGATGGTAGCTATCGTAGCGAAGAGC'L1GAATCT GG’.1 CAT CAT-TGGCCCGAGCGCAGACATTATC CAAT "1 CGAGTGCAAGAGC' 'T' ‘GGTCACGAGGTTCTGAATCTGACCCGCAA1 GGCTATGGTAGCACCCAG TACATTCGTTTTTCGCCGGATTTr1ACCTTCGGCTr1 T GAAGAGAGCCTGGAGGTTGATACCAATCCGTTG CTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCAC CGCC"1GTACGGCATF1GCCATCAACCCAAACCGTGTGT"1CAAGGTTAA"1ACGAA"1 GCATACTACGAGATG AGCGGCCTGGAAG"1CAGCTTCGAAGAACTGCGCACCT"1CGGTGGCCATGACGC'-'AAA'_'T CATT GACAGC 40 TTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATr-1CAAAGACAT"1 GCAAGCACG' " 'GAACAAG AGCATCGTTGGTAC"1ACCGCGTCGTTGCAGTATATGAAGAA1GTGTTTAAAGAGAAGTACCTG CTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTG ACCGAGATTTACACCGAGGACAAC"1TTGTGAAATTCTTCAAAGT G"1 T GAAT CGTAAAACCTATCTGAAT TTTGACAAAGCGGT"1 TT_CAAGATTAACATCGTGCCGAAGGT GAAC"-1ACACCATCTATGACGGTTTTAAC 45 CTGCGTAACACCAACCTGGCGGCGAACT' "-'AACGGTCAGAATACGGAAATCAACAACATGAATTTCACG 1GAAGAACTTCACGGGTCTGTTCGAGT"1CTATAAGCTGCTGr1GCGTGCGCGGTATCATCACCAGC AAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGG GAr'Icr1 GTTCTTTTCGCCAT CCGAAGATAATTTTACCAACGACCTGAACAAGGG"1 GAAGAAA"1 CACCAGC GATACGAATA'L1 T GAAGCAGCGGAAGAGAA"-1ATCAGCCT GGA"-1CTGA"-1 CCAGCAGTACTATCTGACCTT-T 50 1 CGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACA'-"-'A"1 C GGT CAGCTGGAA CTGA"1 GCCGAATAT CGAACGTTTCCCGAACGGCAAAAAG"1ACGAGCTGGACAAGTACACTATGTTCCA"1 CGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCC CTGC"1GAACCCGAGCCGTGTCTATACCTTCT"1 CAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACT GAGGCCGCGATGT'L1 C CT GGGCT GGGTGGAACAGCT GG"_1A"-1ATGAC"-1 T CGAGACGAGCGAAGTG 55 AGCAC"1ACCGACAAAAT-TGCTGATATTACCATCATTAr1 C CCGTATAT CGGCACTGAACATTGGC AACA"1 GCTGTACAAAGACGATTTTGTGGGTGCCCT GAr1C"1T CTCCGGTGCCGTGATTCTGCTGGAGTTC ATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTG ACGG"1T CAGACCAT CGATAACGCGCTGTCGAAACGTAATGAAAAA"1GGGACGAGGTTTACAAATACATT CGAATTGGCTGGCGAAAGTCAATACCCAGA"1CGACCTGATCCGTAAGAAAA"1GAAAGAGGCGCTG 60 T-CAGGCGGAGGCCACCAAAGCAAT"1ATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAG [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar AATAACA'1TAAC1111 CAAF1ATCGATGATTTGAGCAGCAAGCTGAA"YHVV1CTATCAACAAAGCGATGATC AA11A11CAACAAG1111T111:GAATCAGTGTAGCGTTTCGTACC"1GATGAATAGCATGATTCCGTAC1GGCGTC AAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGT'.1GCTGAAAIACATTTACGACAATCGTGGT ACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAA1ACCCTGAGCACCGACATCCCATTT CAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATC AATACTAGCATTCTGAACCTGCGTTACGAGAGCAAGCA"1CTGATTGATC"1GAGCCGTTATGCTAGCAAG ATCAACATCGGTAGCAAGGTCAAF1TTTGACCCGATCGA"1AAGAACCAGATCCAGCTGTTTAA"1CTGGAA TCGAGCAAAAJTGAGGTTATCCTGAAAAAGGCCATTGC1CTACAACTCCAZ1GTACGAGAATTTCTCCACC AGCTTCTGGATTCGCATCCCGAAATACTTCAACAAQN1TAGCCTGAACAACGAGTATACTAT1CATCAAC TGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGAC ACCAAAGAGA'1CAAGCAGCGCGTCGTGTTCAAGTAC'1CTCAAATGATCAACATTTCCGATTACATTAAT CGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAAGAGCAAGATTTACATCAATGGTCGCTTG CAGAAACCGATTAGCAACCTGGGTAA1AT CCACGCAAGCAACAAGATTATGTTCAAAT'.1GGAC GGTTGCCGCGATACCCATCG'" 'ATATCTGGATCAAGF1ATTTCAACCTGTTTGATAAAGAACTGAAJGAG AAGGAGAr1CAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTF1CTGGGGCGATTAT CTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAAT GTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTG AACTCTAGCCTGTACCGTGGTACGAAAT-TCATCATTAAGAAATA".1GCCAGCGGCAACAAAG TAACATT GTGCGTAATAACGAT CG".1GTCTACATCAACGTGG"1 CGTGAAGAA1AAAGAGTACCG'1cr1GGCGACCAAC GCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGF1TGGAGATCCC' ‘GA 'GTCGGTAATCTGAGCCAA GTTATGAAGAGCAAGAACGACAAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAAC AATGGTAACGACATCGGCTTTATTGGTTTCCACCAGF1T CAACAATAT "1Gcr'lAAACTGGTAGCGAGCAA"_‘ TGGTACAA1CGTCAGATTGAGCGCAGCAGCCGTACTC1TGGGCTGTAGCTGGGAGTTTAZ1CCCGGTCGAT GATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 4. ered BoNT/Al "Cat-A" amino acid sequence.
MPFVVKQFNYKD ?VVGV DIAYIKIPNAGQMQPVKAF {:HNKIWVIPERDTFTNPEEG )LNPPPEAKQV.D TYLSTDWEKDNYLKGVTKLFERIYSTDLGR LLTSIVRGI?FWGGSTIDTE_LKVIDTNCIWV: QPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL'1RNGYGSTQYIRFSPDFTFGFEESLEVDTVP __| 3O LGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVN1NAYYEMSGLEVSFEELRC1FGGHDAKFIDS LQENLFR.YYYN {FKDIASTLNKAKSIVGTTASLQYWKNVFKEKYLLSEDTSGKFSVJKL {FDKLYKM.4 TE YTEDNFVKFFKVLN QKT LNFD {AVFKINIVPKVNYTIY DGFNLRNTNLAANFNGQNTEINNMVFT :TSKTKSLDKGYNKALVDLCIKVNWWDLFFSPSEDNFTND_LNKGEEITS DTNI .IQQYY.TFNF ISIENLSSDIIGQ .E .MPNIERFPNG {KYELDKYTWFH YLRAQEFEHGKSRTALTNSVNEALLNPSRVYTFFSSJYVKKVNKN.1bAAMbLGWVbQLVY3bTDbTSbV STTD ' "GNMLYKDDFVGALIFSGAVILL.EFIPEIAIPVLGTFALVSYIANKVL (WDEVY{YIVTVWLAKVNTQID_JIRKKM {EALENQA 1A' KAIIVYQYNQYTEEEK NNIVTNIDDLSSKLVESINKAMIN:VKFLNQCSVSYLMNSMI?YGVKRLEDFDASLK3AL_LKYIYDVRG {DKVNVTLSTDIPFQLSKYVDNQRLLS'1FTEYIKNIINTSI S {HLIDLSRYASK 4O iV-flSSKI MVILKKAIVYNSMYEVFSTSFWIRIPKYFNKIS.
LQDTKEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRL ?ISVLGNI {ASNKIMF M PFVNKQFNYKD PVNGV )IAYI KI PNAGQMQPVKAF {I HNKIWVI PERDTFTNPET.G 3T.NPPPTAKQV .3 VSYYDSTYLS"1DNEK DNYLKGVTKLFERIYSTDLGRVILL"1S IVRGI PFWGGSTI DTE.TJKVIDTVCINVT QTDGSYRSEETNTVIIGTSADI IQFECKSFGHEVLNLTRNGYGSTQYIRTSPDFTTGTEESLEVDTNP __| 50 .. GAGKFATDPAVT .AHF‘. .IHAGHR..YGIAINPNRVFKVN"1NAYYEMS GLEVS RTFGGHDAKFI DS AQENEFRAYYYNKFKDIASTANKAKSIVGTTAS LQYMKNVFKEKYLLSEJTSGKFSVJKLKFD_ T .1.IYTEDNFV.
TVQT I DNALSKRNEKWD.EVY SEQ ID NO: 7. Engineered BoNT/Al "Cat-C" nucleic acid sequence .
ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACA"CAAG ATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTT"1 TAAGAT CCACAACAAGA"1TTGGGTTATCCCG GAGCGTGACACCTTCACGAACCCGGAAGAAGGCGAT C".1 GAACCCGCCACCGGAAGCGAAGCAAG".1CCCT GTCAGCTACTACGAT "1 CGACGTACCTGAGCACGGATAACGAAAAAGATAACTACC"1GAAAGGT G".1GACC AAGCTGTTCGAACGTAT C"1ACAGCACGGA"1C"1GGGTCGCATGCTGCTGACTAGCA"1T GTTCGCGGTATC TGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATT CAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGG"1 CAT CAT "1GGCCCGAGCGCAGACA"1TATC CAATTCGAGTGCAAGAGCTTTGG"1CACGAGGTTCTGAAT C"1 GACCCGCAA"1GGCTA"1GGTAGCACCCAG TACAT'-‘ CGT'-'TTTCGCCGGA".1TT"1ACCTTCGGCT".1 T GAAGAGAGCCTGGAGGT-"1 GA".1ACCAATCCGTTG CTGGGTGCGGGCAAA"1T CGCTACCGATCCGGCT G"1CACGCTGGCCCA"1GAAC"1GATCCACGCAGGCCAC CGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAA"1ACGAATGCATACTACGAGATG AGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGC T "1GCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAAT"1 CAAAGACAT"1GCAAGCACGTTGAACAAG GCCAAAAGCATCGTTGGTACTACCGCGT CG".1TGCAGTATA".1 ".1G'-'GT".1'-‘AAAGAGAAGTACCTG CTGTCCGAGGATACC"1CCGGCAAGTTTAGCG"1T GATAAGCTGAAGTT "1GACAAACTGTACAAG "1-GCT G ACCGAGATTTACACCGAGGACAACTT"1GTGAAATTCTTCAAAGTGTTGAA"1 CGTAAAACCTATCTGAAT TTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAAC CTGCGTAACACCAACCTGGCGGCGAACTT"1AACGGTCAGAA"1ACGGAAN1 CAACAACA"1 GAAT "1 "1 CACG AAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGC".1GCTGTGCG"1 TATCATCACCAGC AAAACCAAAAGCC".1GGACAAAGGC".1ACAACAAGGCGCTGAA"1GACCTGTGCA' "-'AAGG"1AAACAATTGG GATC"1GTTCTTT"1CGCCATCCGAAGA' 'AAT T' ACGACCTGAACAAGGGTGAAGAAATCACCAGC GATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTT AACTTCGACAATGAACCGGAGAACAT"1AGCA"1TGAGAAT C"1 GAGCAGCGACATTA"1CGGTCAGCTGGAA 3O CTGATGCCGAATATCGAACGT"1TCCCGAACGGCAAAAAG".1ACGAGC".1 GGACAAGTACACTATGTT CCA"._‘ CGTGCACAGGAGTT"1 GAACACGG".1AAAAGCCGTA".1 CGCGCTGACCAACAGCGTTAACGAGGCC CTGCTGAACCCGAGCCGTGTC"1A' ‘ACCTTCT' ‘CAGCAGCGAC' 'A' 'GT' AA‘GAAAG"1 GAACAAAGCCAC"1 GAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTG AGCACTACCGACAAAA"wwGerGAmAnTACCAmCAmTAmCCCGnAnAmr1GGTCCGGCACTGAACA'" 'GGC AACATGCTGTACAAAGACGAT"_ .1TTGTGGGTGCCCT GATCT".1CTCCGG".1GCCGTGA".1T CTGCTGGAGTTC ATTCCGGAGATTGCGA".1CCCGGT GT'-'GGG'-'ACC'i'TCGCGC".1 GGT GT CCTACA".1CGCGAATAAGG".1r.1CTG ACGGT"1CAGACCAT CGATAACGCGCTGTCGAAACGTAATGAAAAA"1GGGACGAGG"1TTACAAATACATT GTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTG GAGAA"1 CAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAG 4O AATAACATTAACTTCAA"1AT CGATGATT "1GAGCAGCAAGC"1 GAA"1 GAAT CTA"1 CAACAAAGCGATGATC AATATCAACAAGT-"1TTTGAATCAGTGTAGCGTTTCGTACC"1 GAT GAA".1AGCATGA"1T CCGTATGGCGTC CTGGAGGAC"1TCGACGCCAGCC"1GAAAGATGCGTTGCTGAAATACA"1TTACGACAATCGTGGT ACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTT CAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATC 45 AGCA"1 T CT GAACCT GCGTTACGAGAGCAATCAT C"1 GATTGATC"1 GAGCCGTTATGCTAGCAAG ATCAACATCGGTAGCAAGGTCAA"_1"_1TTGACCCGA"_1 C GATAAGAACCAGATCCAGC".1GTTTAATCTGGAA TCGAGCAAAA"1TGAGGTTATCCTGAAAAAGGCCA"1 T GT CTACAACT CCA".1GTACGAGAAT "1T CTCCACC TGGA"1 T CGCAT CCCGAAATACTTCAACAAGATTAGCCTGAACAACGAGTATACTATCATCAAC TGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGAC 50 ACCAAAGAGA"1CAAGCAGCGCGTCG"1GTTCAAGTACTCTCAAATGA"1CAACAT"1TCCGATTACA'" AA'T CGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAAGAAGAGCAAGAT-TTACA".1CAATGGTCGCTTG ATCGATCAGAAACCGATTAGCAACC".1GGGTAA".1AT CCACGCAAGCAACAAGA' u_.A"1GTTCAAATTGGAC GGTTGCCGCGATACCCATCG"1r-IAr1ATCTGGATCAAG"1ATTTCAACCTGTT"1 GATAAAGAACTGAATGAG AAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTAT 55 CTGCAATACGATAAGCCGTAC"1ATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAA"1AA"_‘ GTGGGTAT".1 CGT AT G".1ATTTGAAGGGTCCGCGTGGCAGCGT".1ATGACGACCAACA".1TTACCTG AGCCTGTACCGTGGTACGAAAT".1 CATCATTAAGAAATA".1 GGCAACAAAGATAACAT-"1 GTGCGTAATAACGATCGTGT C"1ACATCAACG' 'GG' ' C GT GAAGAA"1AAAGAGTACCGT C"1 GGCGACCAAC GCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATC 6O EDTGGT"1ATGAAGAGCAAGAACGACAAGGGTA"1 CACTAACAAGTGCAAGATGAACC"1 GCAAGACAACGTAACGACATCGGCTTTATTGGTT".1CCACCAGT".1 CAACAA"1AT".1GCTAAACTGG".1AGCGAGCAAT [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar ed set by wilksar [Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar TGGTACAATCGTCAGAT"1GAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGAT GATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 8. Engineered BoNT/Al " amino acid sequence.
FNYKD PVNGVDIAYIKIPNAGQMQPVKAF {I HNKIWVI PERDTFTNPEE.G )TNPPPEAKQV _D VSYYDSTYLSTDNEKDNYLKGVTKLF.ERIYSTDLGRVILL"1S IVRGI ?FWGGSTI DTE._.KVIDTNCINVI RSEETNWVI:GPSADIIQFECKSFGHEVLN.4TRNGYGSTQYI RES PDFT FGFEESLEVDTNP .4 .4 GAGKFATDPAVT.JAHELIHAGHR.JYGIAINPNRVF{VN"1NAYYEMSGLEVS FEE._.RTFGGHDAKFI DS QENEFRAYYYNKFKDIASTENKAKSIVGTTASLQYMKNVFKEKYLASEJTSGKFSVDKLKFDKLYKMA wuxvu_.I YTEDNFVKFFKV.JNRKTYLNFD.{AVFKINIVPKVNY"1IYDGFNERNTNLAANFNGQNTEINNWNFT.FEFY <'.LCVRGI ITSKT (SLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDENKGE.LITSTNIEAAEEVI S'DTCQQYY.TFNFDNEPENISIENLSSDIIGQTEVMPVIERFPNGKKYELDKYTMFH.JRAQEFEHGKS RIA EALINPSRVYTFFSSDYVKKVNKAT *.AA\’IJ: T.GWVF.Q .VY DJ: TDETS .LV STTDKIADITIIIPYIGPALNIGNMLYKDDFVGA._.I FSGAVILLEFI PEIAI PVLGT FALVSYIANKV._l TVQT I DNALSKRNEKWDEVY {YIVTNWLAKVNTQT D._;I RKKMKEALENQAEATKAI INYQYNQYTEEEK NNINFNI DD._.SS {LNES INKAMININKFLNQCSVSY.JMNSMI.3YGVKRLEDFDASLK DALEKYIY )NRG LI GQVDQLKDKVNNTLSTDI PFQLSKYVDNQRLLSTFTEYI"(NI I\ITS ILN.JRYESVHL" CMENNS GWKVS L\IYGEI IWTLQDTKEI KQRWFKYSQMINI S DYINRWI FVT ITNNR.
I JQKPI SNLGNI:IASNKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSN LQYDKPYYM.JNLYDPNKYVDVNNVGI RGYMYLKG MTTNI YLNS S LYRGTKFI I KKYAS GN {DNI V QNNDRVYINV V VKNKEYRLATNASQAGVEKI LSALEI PDVGNLSQVVVMKSKNDKGITNKCKMNLQDN NGNDI GFI GFHQFNNIAKLVASNWYNRQI ERS S WEFI PVDDGWGERP.A SEQ ID N019 Engineered BoNT/Al "Cat-D" nucleic acid sequence.
A"-1 GCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAG ATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGAT-"1TGGGTTATCCCG GAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCT GTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACC 3O AAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGAC"1AGCATTGTTCGCGGTATC CCGTTCTGGGGTGGTAGCACGAT_"1GACACCGAAC'L1 GAAGG"-1 TAT CGACACTAACTGCATTAACGTTAT-"1 CAACCGGATGGTAGCTATCGTAGCGAAGAGC"1 GAAT CT GG".1 CAT CAT "1GGCCCGAGCGCAGACATTAT C CAAT "1CGAGTGCAAGAGC' 'TTGG' ‘CACGAGG' ..—.C. 'GAATCTGACCCGCAA"1 GGCTATGGTAGCACCCAG TACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTG C"1GGGTGCGGGCAAA"1T CGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCAC CGCCTGTACGGCA'L1T GCCATCAACCCAAACCGTGTGTTCAAGGTTAA"_1ACGAA"-1 GCATACTACGAGATG AGCGGCCTgGAAG".1CAGCTTCGAAGAACTGCGCACCTTCGGTGGCCA"1GACGC".1AAATTCATTGACAGC TTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACAT"1 GCAAGCACG"1"1GAACAAG GCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTG 4O C"1GTCCGAGGATACCTCCGGCAAGTT"1AGCGTTGATAAGC"1 GAAGT "1 "1GACAAACTGTACAAG "1 GCT G ACCGAGATTTACACCGAGGACAACT"1"1G"1GAAATTCTTCAAaGTGTTGAA"1 C Gr'lAAAACCTATCTGAAT TTTGACAAAGCGG".1 T-"1T CaAGATTAACA".1 C GTGCCGAAGGTGAACTACACCATCTATGACGGT"1T-"1AAC C"1GCGTAACACCAACCTGGCGGCGAACT"1TAACGGTCAGAA"1ACGGAAATCAACAACATGAAT "1T-CACG AAGTTGAAGAACTTCACGGGTCTGTTCGAGT"1CTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGC 45 AAAACCAAAAGCC"1GGACAAAGGCTACAACAAGGCGC"1GAA"1 GACCT GTGCATTAAGGTAAACAA"1"1GG GATCTGTTCTTTTCGCCATCCGAAGA"1AATT AACGACCTGAACAAGGG"1 GAAGAAATCACCAGC GATACGAATATTGAAGCAGCGGAAGAGAA"-1A"-1CAGCC'-' GGA'-'CT GA"-1 CCAGCAGTACTATCTGACCTT-"1 AACTTCGACAATGAACCGGAGAACAT"1AGCA"1T CTGAGCAGCGACA' 'ATCGGTCAGCTGGAA CTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAG"1ACGAGCTGGACAAGTACACTATGTTCCAT 50 TACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCC CTGC"1GAACCCGAGCCGTGTCTATACCTTCT"1 CGACTATGT "1AAGAAAGTGAACAAAGCCAC"1 GAGGCCGCGATGTTCCZ1 GGGCT GGG'JGGAACAGCT GG'-'A"-1A'l' GAC‘_' T CACGGACGAGACGAGCGAAGT G AGCACTACCGACAAAa"1 "1-GC"1GAT "1 GGT CTGAACATTGGC aTTACCATCATTATCCCGTATAT GCTGTACAAAGACGATTTTGTGGGTGCCC' .GAMC. ‘TC' 'CCGG' ‘GCCGTGA' ‘T C' 'GCTGGAGTTC 55 ATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTG ACGGTTCAGACCA"1CGAnAACGCGCTGTCGAAACGTAATGAAAAA"1 GGGACGAGGTTTACAAATACATT GTTACGAATTGGC'L1GGCGAAAGTCaA"-1ACCCAGA"-1CGACCTGATCCGTAAGAAAA"_1GAAAGAGGCGCTG GAGAA".1 CAGGCGGAGGCCACCAAAGCAA"1TATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAG AATAACATTAACT"1 CAATAT CGATGA"1T"1GAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATC 6O ADTCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTC ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r AAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGT ACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTT GAGCAAGTATGT-TGATAA1CAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATC AATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATtGATCTGAGCCGTTATGCAAGCAAG ATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAA TCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCAC AGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAA TGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGA'JCATL'TGGACCTTGCAGGA ACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACA""TCCGATTACATTAA CGTTGGATCTTCGTGACCATTACGAATAACCGTCT AATAACAGCAAGATTTACATCAATGGTCGCTT ATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGA GGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGA AAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTA CTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAA GTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTG AACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATT GTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGCGTAAAGAGTACCGTCTGGCGACCAAC GCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTCGTGTCCGTCGTCZ1GAGCCAA GTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACCGr1 AACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATAT"GCTAAACTGGTAGCGAGCAAF1 TGGTACAATCGTCAGATTGAGCGCCGTAGCCGTCGTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGAT GATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 10. Engineered l "Cat-D" amino acid sequence.
PFVNKQFNYKDPVNGVDIAYTKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGJLNPPPEAKQV D VSYYDSTYLSTDVEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTEJKVIDTNCINVI Q?DGSYRSE34NLVIIGPSADIIQF.3CKSFGHEVLN_JTRNGYGSTQYIRFSPDFTFGFEESLEVDTNP .4 LGAGKFATD?AVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKTIDS1 .QEVEFR.YYYN{F(DIAST_4NKAKSIVGTTASLQYWKNVFKEKYLLSEDTSGKFSV DKL Example 1 Three different examples of an engineered l molecule ing to the present invention were produced.
The amino acids chosen for ation (mutation sites) were selected using a number of different criteria.
The criteria for residue replacement were as follows: 1. Type of residue; 2 Degree of surface exposure; 3 Location with regard to secondary/tertiary structure; 4. Location with regard to the known functional domains of BoNT; Degree of sequence conservation across the BoNT/A or BoNT/E subtypes; 6 Probability of introducing an additional ubiquitination site.
In this e; asparagine (Asn; N) and glutamine (Gln, Q); were selected for on because they are polar, are similar in size to Lys, only form weaker dipole interactions with other residues, and 14% of the molecule is composed of these two residues.
Asn and Gln residues visible at the surface of the molecule were identified from the l structure of BoNT/Al (PDB ID: 3BTA). This s that all replaced residues will be able to display their charge externally. From this list, it was possible to rule out those less suitable for replacement by applying points 3-5 of the selection criteria above (a reiterative process).
Non-conserved residues within BoNT/Al were identified from alignments with other stes of BoNT/A and of the functionally similar BoNT/E serotype. Those that [Annotation] r None set by r [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar appeared as a basic residue in the other sequences were selected as top candidates for replacement.
After successive iterative rounds through the ion criteria mentioned in the sections above, a final list of candidate residues was identified. These were screened for the potential tion of additional ubiquitination consensus ces (using the CKSAAP_UbSite server). The few that were identified were d by changing the replacement lysine to an arginine.
The final e cationic constructs synthesised for BoNT/Al sequence are listed below, named Cat-A, Cat-B and Cat-C. Each construct had a molecular weight of 149,637 s.
Cat-A: N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N886K.
Cat-B: N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N954K.
Cat-C: N930K, S955K, Q991K, N1026K, N1052K, Q1229K, N1025K.
Example 2 The BoNT/B, F and E amino acid sequences were assessed for potential residues that could be substituted with Lys or Arg. This initial assessment identified residues that could be substituted to yield a BoNT/B, E or F protein with an increased pI.
The primary sequence of BoNT/B (Ac: P10844), BoNT/E (Ac: Q00496), and BoNT/F (Ac: P30996) was analysed, and the amino acid composition summarised in the table below: TABLE 3: Serotype Theoretical pI Net charge at pH 7.4 No. Asn & Gln No. Asp & Glu BoNT/B 5.3 -23 179 156 BoNT/E 6.2 -7 160 132 BoNT/F 5.4 -22 169 161 ation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar ation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar From the table, a similarly large number of polar Asn/Gln residues were t in the amino acid sequence as observed for BoNT/Al. There were also a relatively large number of acidic (Asp/Glu) residues which could potentially be changed to either their corresponding neutral residues (Asn/Gln) or to basic residues (Lys or Arg).
Example 3 Identification of preferred clostridial toxin amino acids for modification. ength ural data was available for BoNT/A, BoNT/B, and BoNT/E; however, for the remaining four serotypes, a theoretical model was generated based on ce and associated structural homology using the LOOPP computer program.
Each structure was analysed by AreaIMol (CCP4 suite), and exposed residues were identified as having a sum value greater than 40. Residues with polar-side chains were selected from this list, and from this preference was placed on residues that were either acidic (Asp & Glu) or had an H—bond acceptor side chain (Asn & Gln). The final computational step involved selecting residues in between d—helices and B- strands based on analytical data from the Stride . The structure of each molecule was Visually inspected to identify residues located within ace regions — these were avoided.
For BoNT/Al, the list of preferred residues was supplemented with fianctionally non- conserved residues in at least 90% of all aligned sequences [large non-polar side chains (Met, Pro, Phe, Trp) were considered to be equivalent, small non-polar side chains (Gly, Ala, Val, Leu, 116) were considered to be equivalent, acidic side chains (Asp, Glu) were considered to be lent, and basic side chains (Arg, Lys) were considered to be equivalent]. More specifically, those non-conserved residues that ed as basic residues in at least 10% of the sequences and non-conserved Asn, Gln, Asp or Glu in the reference sequence, were selected as candidates.
For the remaining serotypes, multiple sequence alignments between subtypes was performed to fy functionally nserved residues that appeared as basic rues in at least 10% of the sequences.
[Annotation] wilksar None set by wilksar ation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar Preferred clostridial toxin amino acids for modification: BoNT/A: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086.
BoNT/B: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252, ASN 1273.
BoNT/C1: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, ASP 1081.
BoNT/D: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, GLU 1066, GLN 1122.
BoNT/E: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140.
BoNT/F: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, @1040, ASP 1045, ASN 1055, ASP 1056.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r BoNT/G: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, ASN 1090.
TeNT: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, ASN 1097.
Sequences used Accession numbers: Bo\T/A: P10845 Bo\'T/B: P10844 Bo\T/C1: P1864O BoVT/D: P1932] BokT/E: Q00496 : YP_001390123 BoNT/G: Q60393 TeNT: PO4958 Structural data source Crystal structures of BoNT/A (3BTA.pdb), BoNT/B , and BoNT/E (3FFZ.pdb) obtained from RCSB.
Homology modelling of BoNT/Cl, BoNT/D, BoNT/F, BoNT/G, and TeNT performed using LOOPP and the following sequences, respectively: P18640, P19321, YP_001390123, Q60393, and P04958.
Structural analysis 3O d residues determined using AreaIMol from the CCP4 suite. ary structure assignments determined using .
Interface residues determined by Visual inspection with RasMol.
[Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by r [Annotation] wilksar None set by wilksar [Annotation] wilksar ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar Sequence analysis Full-length BoNT sequences obtained from NCBI.
Alignments performed with ClustalX.
Example 4 Cloning, Expression and Purification DNA constructs encoding the engineered BoNT/A molecules described in Example 1 were synthesised, cloned into the p140] expression vector and then transformed into BL21 (DE3) E. coli. This allowed for soluble over-expression of the inant Cat- A, Cat—B and Cat-C proteins in BL21(DE3) E. coli.
The recombinant engineered BoNTs were purified using classical chromatography techniques from the E. coli lysates. An initial ation step using a cation- exchange resin was employed, followed by an intermediate ation step using a hydrophobic interaction resin. The recombinant ered BoNT single-chain was then cleaved by proteolysis, ing in the activated di-chain engineered BoNT. A final purification step was then employed to remove remaining contaminants.
Example 5 Characterization of purified engineered BoNTs The engineered BoNTs described in e 1 above were characterised experimentally as follows.
Measurement of the p1 showed that the engineered BoNTs had an isoelectric point greater than that of unmodified (native) BoNT/Al — see Figure l and Table below.
[Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar TABLE 4: BoNT/Al le pI pI ved) (calculated) Engineered, "Cat-A" 6.9 ~8.0 [Cat5v2(K1064H/N886K] Engineered, "Cat-B" 6.9 ~8.0 [Cat5v2(K1064/N954K)] Engineered, "Cat-C" 6.9 7.8-8.0 [Cat5v2(K1064H/N1025K)] Native BoNT/Al 6.05 ~7.4 [rBoNT/Al] The ability of the engineered BoNTs to enter neurons and cleave SNAP-25 (the target ofBoNT/Al) was assessed using rat embryonic spinal cord neurons (eSCN). Figure 2 shows that the ered BoNTs retained the same ability to enter the neuron and cleave SNAP-25 as native BoNT/Al.
Potency of the engineered BoNTs was further ed using the mouse phrenic nerve hemi-diaphragm assay (mPNHD). Figure 3 shows that the engineered BoNTs retained the same ability to inhibit the contractile abilities of the mouse iaphragm as native BoNT/A1.
The in vivo mouse Digital Abduction Score (DAS) assay was used to assess potency as well as safety relative to native BoNT/Al. Both molecules displayed a higher safety ratio relative to native BoNT/Al and were slightly more . These data are presented below (Table 4).
[Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar TABLE 4: le DAS EDso Dose DAS 4 Dose for -10% Safety Ratio (pg/mouse) (pg/mouse) ABW (pg/mouse) Native 2 10-20 9.9- 14.5 7 BoNT/Al (n=5) Engineered, 1.16 10-20 27.4 24 "Cat-A" ered, 1.79 25 47.6 27 "Cat-B" -DAS ED50: Calculated dose inducing a DAS 2 -Dose DAS 4: Experimental dose inducing a DAS 4 -BW: Body weight -Dose for —10% ABW: Calculated dose inducing a se of 10% on BW in comparison to BW at D0 -Safety Ratio: Dose for -10% ABW / DAS ED50 The Safety Ratio is a measure of a negative effect of BoNT treatment (weight loss) with respect to potency (half maximal digital abduction score (DAS)). It is calculated as the ratio between -10% Body Weight (BW) and the DAS ED50, where -10%BW refers to the amount of BoNT (pg/animal) required for a 10% decrease in body weight, and ED50 refers to the amount of BoNT (pg/animal) that will produce a DAS of 2.
The DAS assay is performed by injection of 20111 of engineered clostridial toxin, formulated in Gelatin Phosphate , into the mouse gastrocnemius/soleus complex, followed by assessment of Digit Abduction as previously reported by Aoki (Aoki KR, Toxicon 39: 820; 2001).
Example 6 Ether engineered clostridial toxin according to the present invention was designed [Annotation] wilksar None set by r [Annotation] r ionNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar using the ia as set out in Example 1 above.
This cationic construct was also d from BoNT/Al and had a calculated p1 of 7.4, and a molecular weight of 149,859. The construct was named Cat-D. While ucts Cat-A, Cat-B and Cat—C comprised residues mutated to lysine, Cat-D comprised residues mutated to arginine.
Cat-D: N1188R, D1213R, G1215R, N1216R, N1242R, N1243R, SlZ74R, T1277R.
Example7 Treatment of a patient suffering from Cervical Dystonia A 50 year old female ing from spasmodic torticollis presents in the clinic, having previously been treated with a eutically effective amount of a standard BoNT/A preparation into the neck muscle; however, the patient experienced dysphagia due to spread of the toxin into the oropharynx. The patient is treated with an injection in the neck muscles of approximately 1.5ng (or more) of an engineered BoNT/A of the present invention. The patient’s torticollis is significantly improved after 3-7 days, without the development of dysphagia, and the patient is able to hold her head and shoulder in a normal position for at least five months. Due to the engineered BoNT/A molecule’s enhanced tissue retention and reduced spread, the physician is able to inject more product without fear of side effects; the enhanced dose leads to an extended duration of action.
Example 8 Treatment of a patient suffering from blepharospasm A 47 year old male presents in the clinic with blepharospasm. The patient is treated by injection of between 5 pg and 25 pg of an engineered BoNT/A of the present ion into the lateral rsal laris oculi muscle of the upper lid and the lame—tarsal orbicularis oculi of the lower lid. Alleviation of the patient’s symptoms [Annotation] r None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] r ed set by wilksar occur in about a week and last at least five months, without eye ptosis. The increased safety of the polypeptide of the t invention allows the ian to increase the dose and therefore extend the duration of the clinical effect.
Example 9 A 27 year old male suffering from cerebral palsy presents at the clinic with debilitating equinus foot and has difficulty walking. The patient had previously been treated with a therapeutically ive amount of , where alleviation improvement in his gait was accompanied by muscle weakness and pain in his extremities. The patient is treated by injection of about 20 pg/kg of an engineered BoNT/A of the present invention into each of two sites in the medial and lateral heads of the gastrocnemius muscle of the affected lower limb(s). Within a week, the patient’s gait improves without previously seen side effects and the symptomatic relief lasts for at least four months. The ability to dose higher amounts of drug product leads to treatments which result in an extended duration of action. 1003417369
Claims (35)
1. An engineered clostridial toxin comprising between 4 and 40 amino acid modifications d in the clostridial toxin HCN domain, wherein said between 4 and 5 40 amino acid modifications increase the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of an otherwise identical clostridial toxin g said between 4 and 40 amino acid modifications, wherein the engineered clostridial toxin is: a. an engineered botulinum neurotoxin A (BoNT/A) having a pI of at least 10 6.6 and the n 4 and 40 amino acid modifications comprise modification of at least 4 amino acids ed from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 15 1080, GLU 1081, GLU 1083, and ASP 1086; b. an engineered BoNT/B having a pI of at least 5.5 and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 20 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, and GLU 1072; c. an engineered BoNT/C1 having a pI of at least 5.7 and the between 4 and 40 amino acid cations comprise modification of at least 4 amino acids selected from: ASN 881, ASP 898, GLU 916, GLU 927, 25 ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081; d. an engineered BoNT/D having a pI of at least 5.7 and the between 4 and 40 amino acid modifications comprise modification of at least 4 30 amino acids selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, and GLU 1066; e. an engineered BoNT/E having a pI of at least 6.2 and the between 4 35 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 1003417369 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, and ASN 1035; f. an engineered BoNT/F having a pI of at least 5.8 and the between 4 5 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056; 10 g. an engineered BoNT/G having a pI of at least 5.4 and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 15 1073, ASN 1075, and ASN 1090; or h. an engineered s neurotoxin (TeNT) having a pI of at least 6.0 and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, 20 ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097; and wherein the between 4 and 40 amino acid modifications are selected from: i. tution of an acidic amino acid residue with a basic amino acid residue; 25 ii. substitution of an acidic amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged amino acid residue with a basic amino acid residue; iv. ion of a basic amino acid e; or 30 v. deletion of an acidic amino acid residue; and wherein the ered clostridial toxin has a Safety Ratio of at least 8, n the Safety Ratio is calculated as: dose of toxin required for -10% bodyweight change measured as pg/mouse divided by DAS ED50 measured as pg/mouse, wherein ED50 = dose required to produce a DAS score of 2.
2. A method for producing an engineered clostridial toxin, the method comprising modifying an HCN domain of a clostridial toxin to introduce between 4 and 1003417369 40 amino acid modifications, thereby producing the engineered clostridial toxin, wherein the between 4 and 40 amino acid modifications increase the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of an otherwise identical clostridial toxin lacking the between 5 4 and 40 amino acid modifications, and wherein the clostridial toxin is a botulinum neurotoxin A (BoNT/A) and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 10 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, and ASP 1086, or wherein the idial toxin is a BoNT/B and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, 15 ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, and GLU 1072, or wherein the clostridial toxin is a BoNT/C1 and the between 4 and 40 amino acid modifications comprise cation of at least 4 amino acids selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, 20 GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081, or wherein the idial toxin is a BoNT/D and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, 25 GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, and GLU 1066, or wherein the clostridial toxin is a BoNT/E and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids ed from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, 30 ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, and ASN 1035, or n the clostridial toxin is a BoNT/F and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: 35 ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056, or 1003417369 wherein the clostridial toxin is a BoNT/G and the between 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 5 1057, GLU 1073, ASN 1075, and ASN 1090, or wherein the clostridial toxin is a tetanus neurotoxin (TeNT) and the n 4 and 40 amino acid modifications comprise modification of at least 4 amino acids selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, 10 ASN 1081, and ASN 1097, and wherein the between 4 and 40 amino acid modifications are selected from: i. substitution of an acidic amino acid residue with a basic amino acid residue; ii. substitution of an acidic amino acid e with an uncharged 15 amino acid residue; iii. substitution of an ged amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid e; and v. deletion of an acidic amino acid residue. 20 wherein the engineered clostridial toxin has a Safety Ratio of at least 8, wherein the Safety Ratio is calculated as: dose of toxin required for -10% bodyweight change measured as pg/mouse divided by DAS ED50 measured as pg/mouse, wherein ED50 = dose required to produce a DAS score of 2. 25
3. The method according to claim 2, wherein the n 4 and 40 amino acid modifications are introduced by: a. synthesising a nucleic acid sequence that s the engineered clostridial toxin comprising the between 4 and 40 amino acid modifications; or b. providing a DNA sequence ng a clostridial toxin and modifying the DNA 30 sequence to introduce the between 4 and 40 amino acid modifications into the encoded engineered clostridial toxin.
4. A method for producing an ered clostridial toxin, the method comprising modifying a clostridial toxin receptor binding domain (HC domain) 35 of a clostridial toxin to introduce at least four amino acid modifications, thereby producing the ered clostridial toxin, 1003417369 wherein the at least four amino acid modifications se the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of the otherwise identical clostridial toxin lacking the at least four amino acid modifications, and 5 wherein the clostridial toxin is botulinum oxin A A) and the at least four amino acid modifications consist of modification of at least four amino acids selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 10 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, or wherein the clostridial toxin is BoNT/B and the at least four amino acid modifications consist of modification of at least four amino acids selected 15 from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252, and ASN 1273, or wherein the clostridial toxin is BoNT/C1 and the at least four amino 20 acid modifications t of modification of at least four amino acids selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081, or wherein the clostridial toxin is BoNT/D and the at least four amino acid 25 modifications consist of cation of at least four amino acids selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122, or wherein the clostridial toxin is BoNT/E and the at least four amino acid 30 modifications consist of modification of at least four amino acids selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140, or 35 wherein the clostridial toxin is BoNT/F and the at least four amino acid modifications consist of modification of at least four amino acids selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, 1003417369 ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056, or wherein the clostridial toxin is BoNT/G and the at least four amino acid modifications consist of modification of at least four amino acids selected 5 from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 1090, or wherein the clostridial toxin is a tetanus neurotoxin (TeNT) and the at least four amino acid modifications t of modification of at least four 10 amino acids ed from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097, and wherein the at least four amino acid modifications are selected from: i. substitution of an acidic amino acid residue with a basic 15 amino acid residue; ii. substitution of an acidic amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged amino acid residue with a basic amino acid e; 20 iv. insertion of a basic amino acid e; or v. deletion of an acidic amino acid residue, and wherein the engineered clostridial toxin has a Safety Ratio of at least 8, wherein the Safety Ratio is calculated as: dose of toxin required for -10% bodyweight change measured as pg/mouse divided by DAS ED50 measured 25 as se, wherein ED50 = dose ed to produce a DAS score of 2.
5. A method for increasing the isoelectric point (pI) of a clostridial toxin, the method comprising modifying a clostridial toxin receptor binding domain (HC domain) of a clostridial toxin to introduce at least four amino acid 30 modifications, thereby sing the pI of the engineered clostridial toxin, wherein the at least four amino acid modifications increase the pI of the clostridial toxin to a value that is at least 0.2 pI units higher than the pI of the otherwise identical clostridial toxin lacking the at least four amino acid modifications, and 35 wherein the clostridial toxin is BoNT/A and the at least four amino acid modifications comprise modification of at least four amino acids selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, 1003417369 SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, or 5 wherein the clostridial toxin is BoNT/B and the at least four amino acid cations comprise modification of at least four amino acids selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, 10 GLU 1072, GLN 1176, GLU 1189, GLU 1252, and ASN 1273, or wherein the clostridial toxin is BoNT/C1 and the at least four amino acid modifications comprise modification of at least four amino acids selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, 15 ASP 1062, ASP 1064, GLU 1079, and ASP 1081, or wherein the clostridial toxin is BoNT/D and the at least four amino acid modifications se modification of at least four amino acids selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, 20 ASP 1033, GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122, or wherein the clostridial toxin is BoNT/E and the at least four amino acid cations comprise modification of at least four amino acids selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 25 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140, or wherein the clostridial toxin is BoNT/F and the at least four amino acid modifications comprise modification of at least four amino acids selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, 30 ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056, or wherein the clostridial toxin is BoNT/G and the at least four amino acid modifications comprise modification of at least four amino acids selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, 35 GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 1090, or 1003417369 wherein the idial toxin is a tetanus neurotoxin (TeNT) and the at least four amino acid modifications comprise modification of at least four amino acids selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 5 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097, and wherein the at least four amino acid modifications are selected from: i. substitution of an acidic amino acid residue with a basic amino acid residue; ii. substitution of an acidic amino acid residue with an 10 uncharged amino acid residue; iii. substitution of an uncharged amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; or v. deletion of an acidic amino acid residue, and 15 wherein the engineered clostridial toxin has a Safety Ratio of at least 8, wherein the Safety Ratio is calculated as: dose of toxin required for -10% bodyweight change measured as pg/mouse d by DAS ED50 measured as pg/mouse, wherein ED50 = dose ed to produce a DAS score of 2. 20
6. The method ing to claim 4 or 5, wherein the at least four amino acid modifications are introduced by: a. synthesising a nucleic acid sequence that encodes the engineered clostridial toxin comprising the at least four amino acid modifications; 25 b. providing a DNA sequence encoding a clostridial toxin and modifying the DNA sequence to uce the at least four amino acid modifications into the encoded engineered clostridial toxin.
7. The method according to claim 3 or 6, n the method further comprises 30 expressing the synthesised nucleic acid or DNA ce in a suitable host cell in vitro, lysing the host cell to provide a host cell homogenate containing the engineered clostridial toxin, and isolating the ered clostridial toxin.
8. The engineered clostridial toxin or method according to any one of claims 1-7, 35 wherein the engineered clostridial toxin comprises an amino acid sequence having at least 90% sequence identity to an amino acid ce selected from SEQ ID NOs: 4, 6, or 8. 1003417369
9. The engineered clostridial toxin or method according to any one of claims 1-8, wherein the engineered clostridial toxin comprises an amino acid ce ed from SEQ ID NOs: 4, 6, or 8.
10.The engineered clostridial toxin or method according to any one of claims 1-9, further comprising at least one amino acid modification d in the HCC domain.
11. The engineered clostridial toxin or method according to any one of claims 1- 10 10, wherein the engineered clostridial toxin is an engineered BoNT/A comprising modification of the ing seven amino acids: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, and GLN 1229.
12. The engineered clostridial toxin or method according to any one of claims 1- 15 10, wherein the engineered clostridial toxin is an engineered BoNT/A comprising modification of the following seven amino acids: ASN 930, ASN 954, SER 955, GLN 991, ASN 1026, ASN 1052, and GLN 1229.
13. The engineered clostridial toxin or method according to any one of claims 1- 20 10, wherein the engineered clostridial toxin is an engineered BoNT/A comprising modification of the following seven amino acids: ASN 930, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229.
14. The engineered clostridial toxin or method according to any one of claims 1- 25 13, wherein the amino acid modifications increase the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.5 pI units higher than the pI of an otherwise identical clostridial toxin lacking said amino acid modifications.
15. The engineered clostridial toxin or method according to any one of claims 1- 30 14, n the amino acid modifications increase the isoelectric point (pI) of the engineered idial toxin to a value that is at least one pI unit higher than the pI of an otherwise identical clostridial toxin lacking said amino acid cations.
16. The engineered clostridial toxin or method according to any one of claims 1- 35 15, wherein said amino acid modifications increase the ctric point (pI) of the engineered clostridial toxin to a value that is at least two pI units higher than the pI of an otherwise identical clostridial toxin lacking said amino acid modifications. 1003417369
17. The engineered clostridial toxin or method according to any one of claims 1- 16, wherein said amino acid modifications increase the isoelectric point (pI) of the engineered clostridial toxin to a value that is between 2 and 5 pI units higher than the 5 pI of an otherwise identical clostridial toxin lacking said amino acid modifications.
18. The engineered clostridial toxin or method according to any one of claims 1- 17, wherein the engineered clostridial toxin has a pI of at least 6. 10
19. The engineered clostridial toxin or method according to any one of claims 1- 18, wherein the engineered clostridial toxin has a pI of between 6 and 10.
20. The engineered clostridial toxin or method according to any one of claims 1- 19, wherein the engineered clostridial toxin has a pI of between 7 and 9.
21. The ered clostridial toxin or method according to any one of claims 1- 20, wherein the engineered idial toxin has a pI of between 8 and 9.
22. The engineered clostridial toxin or method ing to any one of claims 1- 20 21, wherein the amino acid modifications se modification of an amino acid residue selected from: an aspartic acid residue, a glutamic acid e, a histidine residue, a serine residue, a ine residue, an asparagine residue, a glutamine residue, a cysteine residue, or a tyrosine residue. 25
23. The engineered clostridial toxin or method according to claim 22, wherein the amino acid residue is substituted with a lysine residue or an arginine residue.
24. An engineered clostridial toxin comprising SEQ ID NO: 2 that has at least four amino acid modifications located in the clostridial toxin or binding 30 domain (HC domain), n the at least four amino acid modifications increase the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of a clostridial toxin comprising SEQ ID NO: 2 lacking the at least four amino acid cations, 35 wherein the at least four amino acid modifications consist of modification of at least four amino acids selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 1003417369 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, 5 wherein the at least four amino acid modifications are selected from: i. substitution of an acidic amino acid residue with a basic amino acid residue; ii. substitution of an acidic amino acid residue with an ged amino acid residue; 10 iii. substitution of an uncharged amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; or v. deletion of an acidic amino acid residue, and n the engineered idial toxin has a Safety Ratio of at least 15 8, wherein the Safety Ratio is calculated as: dose of toxin ed for - 10% bodyweight change measured as pg/mouse divided by DAS ED50 measured as pg/mouse, wherein ED50 = dose required to e a DAS score of 2. 20 25. The engineered clostridial toxin according to claim 24, wherein the engineered clostridial toxin: a. is encoded by a nucleotide sequence comprising SEQ ID NO: 3 and/or comprises SEQ ID NO: 4; or b. is encoded by a nucleotide sequence comprising SEQ ID NO: 5
25.and/or comprises SEQ ID NO: 6; or c. is d by a nucleotide sequence comprising SEQ ID NO: 7 and/or comprises SEQ ID NO: 8; or d. is encoded by a nucleotide sequence comprising SEQ ID NO: 9 and/or comprises SEQ ID NO: 10.
26. An engineered clostridial toxin comprising at least four amino acid modifications located in the clostridial toxin receptor binding domain (HC domain), wherein the at least four amino acid modifications increase the 35 isoelectric point (pI) of the engineered idial to a value that is at least 0.2 pI units higher than the pI of the otherwise identical clostridial toxin lacking the at least four amino acid modifications, and 1003417369 wherein the engineered clostridial toxin is BoNT/B and the at least four amino acid modifications consist of modification of at least four amino acids selected from: ASN 873, ASN 874, GLU 892, ASP 895, ASN 906, ASP 940, ASN 948, GLU 949, ASN 958, ASN 959, ASN 979, ASN 990, GLU 993, ASP 5 994, GLU 997, ASN 1012, ASN 1019, ASP 1030, ASP 1047, ASP 1049, GLU 1065, GLU 1072, GLN 1176, GLU 1189, GLU 1252, and ASN 1273, or n the engineered clostridial toxin is BoNT/C1 and the at least four amino acid modifications consist of modification of at least four amino acids selected from: ASN 881, ASP 898, GLU 916, GLU 927, ASN 952, ASN 10 964, ASN 965, ASN 984, GLU 985, ASP 986, ASP 996, ASN 1000, GLU 1036, ASN 1041, ASP 1062, ASP 1064, GLU 1079, and ASP 1081, or wherein the engineered clostridial toxin is BoNT/D and the at least four amino acid modifications consist of cation of at least four amino acids selected from: ASN 877, ASP 893, ASN 894, ASN 898, ASN 920, ASN 15 945, ASN 948, GLU 957, GLN 958, ASN 959, ASN 968, ASN 979, GLU 1030, ASP 1031, ASP 1033, GLU 1047, GLU 1051, ASN 1052, GLU 1066, and GLN 1122, or wherein the engineered clostridial toxin is BoNT/E and the at least four amino acid modifications consist of modification of at least four amino acids 20 selected from: ASN 859, ASP 860, ASN 892, ASP 893, ASP 904, ASP 909, ASN 928, ASN 932, ASN 934, ASN 935, GLU 936, ASP 945, ASN 946, ASN 947, ASN 966, ASN 976, ASN 979, ASN 981, ASP 985, GLN 1014, ASN 1019, ASN 1022, ASP 1027, ASN 1035, and ASN 1140, or wherein the engineered clostridial toxin is BoNT/F and the at least four 25 amino acid modifications consist of modification of at least four amino acids selected from: ASN 879, ASP 896, ASN 922, ASN 923, ASN 928, ASN 947, ASN 950, ASN 952, ASN 953, GLU 954, ASN 963, ASN 964, ASN 965, ASN 987, GLN 997, ASN 1037, ASP 1040, ASP 1045, ASN 1055, and ASP 1056, 30 wherein the engineered clostridial toxin is BoNT/G and the at least four amino acid modifications consist of cation of at least four amino acids selected from: ASP 900, ASN 909, ASN 910, GLU 912, ASN 913, ASN 945, ASN 947, GLU 956, ASN 965, ASP 966, ASN 986, ASN 1001, ASN 1038, ASP 1040, ASN 1046, ASP 1057, GLU 1073, ASN 1075, and ASN 35 1090, or n the ered clostridial toxin is a tetanus neurotoxin (TeNT) and the at least four amino acid modifications consist of modification of at 1003417369 least four amino acids selected from: ASN 893, ASP 894, ASP 911, ASN 919, ASN 927, ASN 928, GLU 929, GLN 968, ASN 972, GLU 973, GLU 1010, ASP 1018, ASN 1079, ASN 1080, ASN 1081, and ASN 1097, and wherein the at least four amino acid modifications are selected from: 5 i. substitution of an acidic amino acid residue with a basic amino acid residue; ii. substitution of an acidic amino acid e with an uncharged amino acid residue; iii. substitution of an uncharged amino acid residue with a basic 10 amino acid residue; iv. insertion of a basic amino acid residue; or v. deletion of an acidic amino acid residue, and n the engineered clostridial toxin has a Safety Ratio of at least 8, wherein the Safety Ratio is calculated as: dose of toxin required for - 15 10% bodyweight change measured as se divided by DAS ED50 measured as pg/mouse, wherein ED50 = dose required to produce a DAS score of 2.
27. A nucleic acid comprising a nucleic acid sequence encoding an ered 20 clostridial toxin according to any one of claims 1 or 8-26.
28. An in vitro method of producing a single-chain engineered idial toxin protein having a light chain and a heavy chain, the method comprising expressing a nucleic acid according to claim 27 in a suitable host cell, lysing the host cell to 25 provide a host cell homogenate containing the single-chain engineered clostridial toxin protein, and isolating the -chain engineered clostridial toxin protein.
29. A method of activating an engineered idial toxin, the method comprising providing a single-chain engineered clostridial toxin protein obtainable by the method
30.of claim 28, contacting the polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the light chain and heavy chain, and converting the polypeptide into a in polypeptide wherein the light chain and heavy chain are joined together by a disulphide bond. 35 30. A di-chain engineered clostridial toxin obtainable by the method of claim 29. 1003417369
31. An engineered clostridial toxin according to any one of claims 1 or 8-26 or a di-chain engineered clostridial toxin according to claim 30, for use in medicine.
32. Use of an engineered clostridial toxin according to any one of claims 1 or 8-26 5 or a di-chain engineered clostridial toxin according to claim 30 in the manufacture of a medicament for ting or treating a disease or condition selected from: strabismus, blepharospasm, squint, dystonia, torticollis, neuromuscular disorder or condition of ocular ty, writer's cramp, m, Wilson's e, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity 10 resulting in abnormal r control, animus, back spasm, charley horse, tension headaches, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, ring, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, enic bladder, anismus, limb spasticity, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive tion, excessive 15 gastrointestinal secretions, muscle pain, headache pain, , e disorders, uro-genital disorders, urogenital-neurological disorders, chronic neurogenic inflammation, and a smooth muscle disorder.
33. The use according to claim 32, wherein the dystonia is spasmodic dystonia, 20 oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, or cervical ia, or wherein the torticollis is spasmodic torticollis, or wherein the neuromuscular disorder or condition of ocular motility is concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, or dysthyroid hy, or wherein the muscle pain is pain from muscle spasms.
34. An engineered clostridial toxin according to any one of claims 1 or 8-26 or a di-chain ered clostridial toxin according to claim 30 for use in cosmetic treatment benefitting from cell/muscle incapacitation. 30
35. The engineered clostridial toxin or di-chain engineered clostridial toxin according to claim 34 for use in ng brow furrows and/or skin wrinkles.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB1312317.9A GB201312317D0 (en) | 2013-07-09 | 2013-07-09 | Cationic neurotoxins |
| GB1312317.9 | 2013-07-09 | ||
| PCT/GB2014/052097 WO2015004461A1 (en) | 2013-07-09 | 2014-07-09 | Cationic neurotoxins |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NZ715570A NZ715570A (en) | 2021-04-30 |
| NZ715570B2 true NZ715570B2 (en) | 2021-08-03 |
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