Patent Application
20230038233
The present invention relates to the treatment of neurological disorders.
Neurological disorders include neuronal injuries, neurodegenerative disorders, sensory disorders, and autonomic disorders.
Neuronal injuries, such as spinal cord injuries (SCI), induce degeneration of injured axons preventing normal sensory, motor, and autonomic function. Recovery can occur by endogenous mechanisms such as regeneration of injured axons and the collateral sprouting of undamaged axons, resulting in the reinnervation of denervated targets. However, the regenerative capacity of the injured neurons (especially the spinal cord) is limited in adult mammals and patients can suffer various disabilities which greatly impact quality of life.
Conventional therapeutics for neuronal injuries include interleukin-6 (IL-6) and stem cell transplantation, however few are at a phase of development for use in the clinic. Therefore, there remains a need for a therapeutic for neuronal injuries capable of promoting neuronal growth or repair.
Bacteria in the genus Clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, and X (see WO 2018/009903 A2), as well as those produced by C. baratii and C. butyricum.
Among the clostridial neurotoxins are some of the most potent toxins known. By way of example, botulinum neurotoxins 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 function 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 nervous system.
In nature, clostridial neurotoxins 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. Cleavage 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 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 an N-terminal translocation component (HN domain) and a C-terminal targeting component (HC domain). The cleavage site is located between the L-chain and the translocation domain components. Following binding of the HC 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 non-cytotoxic protease).
Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin). The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell. The protease function 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 neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.
In view of the ubiquitous nature of SNARE proteins, clostridial neurotoxins such as botulinum toxin have been successfully employed in a wide range of therapies.
WO 2016/170501 A1 describes the use of catalytically active full-length BoNT/A (containing the L-chain and complete H-chain including the HN and HC domains) for the treatment of paralysis caused by spinal cord injury. WO 2016/170501 A1 teaches that each of the functional domains of BoNT/A are essential for the therapeutic effects observed, including the H-chain binding and translocation capabilities and the L-chain non-cytotoxic protease activity. As described above, full-length clostridial neurotoxins are extremely potent, necessitating adoption of specific safety procedures when handling the toxin. Moreover, spread 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 neurotoxin therapeutics (such as BoNT therapeutics) at high doses, concentrations and injection volumes. Adverse effects associated with this problem that have been reported for commercial BoNT/A therapeutics include asthenia, generalised muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and breathing difficulties. Swallowing and breathing difficulties can be life threatening and there have been reported deaths related to the spread of toxin effects. Thus, there is a need for a safer therapeutic for promoting neuronal growth or repair.
Given their size, use of the full-length clostridial neurotoxins (˜150 kDa) or complete H-chains thereof (˜100 kDa) is associated with an increased risk of eliciting an immune response in a subject being treated with said polypeptide. Moreover, the presence of the entire H-chain (and in particular the HC domain) results in polypeptide binding to clostridial neurotoxin target receptors, which may be associated with unwanted off-target effects in a subject administered said polypeptide.
The present invention overcomes one or more of the above-mentioned problems.
The present inventors have surprisingly found that a polypeptide comprising a clostridial neurotoxin L-chain and/or a fragment of a clostridial neurotoxin H-chain (e.g. the translocation domain (HN) or the receptor binding domain (HC)) promotes neuronal growth or repair, and thus finds utility in treating neurological disorders. Advantageously, this allows for the use of non-toxic (or substantially non-toxic) fragments of clostridial neurotoxins, which given the smaller size (compared to the full-length H-chain or full-length clostridial neurotoxin), are less likely to provoke an immune response in a subject administered said fragments. Moreover, the non-toxic (or substantially non-toxic) fragments are less expensive and/or less complex to manufacture than full-length clostridial neurotoxins. Additionally, the non-toxic (or substantially non-toxic) fragments constitute a more well-defined therapeutic than the full-length clostridial toxins, and given the shorter length of the polypeptides there is a reduced probability of, for example, cysteine shuffling between domains.
Thus, in one aspect the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises:
In a related aspect there is provided a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises:
In another aspect there is provided use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises:
In one aspect the invention provides a polypeptide for use in treating a neurological disorder in a subject, wherein the polypeptide comprises:
In a related aspect there is provided a method for treating a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises:
In another aspect there is provided use of a polypeptide in the manufacture of a medicament for treating a neurological disorder in a subject, wherein the polypeptide comprises:
In one embodiment a polypeptide of the invention comprises a clostridial neurotoxin L-chain. It is preferred that the L-chain is catalytically inactive.
Thus, in one aspect, the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a catalytically inactive clostridial neurotoxin L-chain.
In a related aspect the invention provides a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises a catalytically inactive clostridial neurotoxin L-chain.
In another related aspect the invention provides use of a polypeptide comprising a catalytically inactive clostridial neurotoxin L-chain in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject.
In one aspect, the invention provides a polypeptide for use in treating a neurological disorder in a subject, wherein the polypeptide comprises a catalytically inactive clostridial neurotoxin L-chain.
In a related aspect the invention provides a method for treating a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises a catalytically inactive clostridial neurotoxin L-chain.
In another related aspect the invention provides use of a polypeptide comprising a catalytically inactive clostridial neurotoxin L-chain in the manufacture of a medicament for treating a neurological disorder in a subject.
The present inventors were the first to show that the catalytic activity of a clostridial neurotoxin L-chain is not necessary to promote neuronal growth or neuronal repair. Thus, the present invention allows for the provision of a safer (less toxic) therapeutic.
Active clostridial neurotoxin L-chain has non-cytotoxic protease activity. Specifically, active clostridial neurotoxin L-chain has endopeptidase activity and is capable of cleaving a protein of the exocytic fusion apparatus in a target cell. A protein of the exocytic fusion apparatus is preferably a SNARE protein, such as SNAP-25, synaptobrevin/VAMP, or syntaxin.
The term “catalytically inactive” as used herein in respect of a clostridial neurotoxin L-chain means that said L-chain exhibits substantially no non-cytotoxic protease activity, preferably the term “catalytically inactive” as used herein in respect of a clostridial neurotoxin L-chain means that said L-chain exhibits no non-cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin L-chain is one that does not cleave a protein of the exocytic fusion apparatus in a target cell. The term “substantially no non-cytotoxic protease activity” means that the clostridial neurotoxin L-chain has less than 5% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain, for example less than 2%, 1% or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain. Non-cytotoxic protease activity can be determined in vitro by incubating a test clostridial neurotoxin L-chain with a SNARE protein and comparing the amount of SNARE protein cleaved by the test clostridial neurotoxin L-chain when compared to the amount of SNARE protein cleaved by a catalytically active clostridial neurotoxin L-chain under the same conditions. Routine techniques, such as SDS-PAGE and Western blotting can be used to quantify the amount of SNARE protein cleaved. Suitable in vitro assays are described in WO 2019/145577 A1, which is incorporated herein by reference.
Cell-based and in vivo assays may also be used to determine if a clostridial neurotoxin comprising an L-chain and a functional cell binding and translocation domain has non-cytotoxic protease activity. Assays such as the Digit Abduction Score (DAS), the dorsal root ganglia (DRG) assay, spinal cord neuron (SCN) assay, and mouse phrenic nerve hemidiaphragm (PNHD) assay are routine in the art. A suitable assay for determining non-cytotoxic protease activity may be one described in Donald et al (2018), Pharmacol Res Perspect, e00446, 1-14, which is incorporated herein by reference.
A catalytically inactive L-chain may have one or more mutations that inactivate said catalytic activity. For example, a catalytically inactive BoNT/A L-chain may comprise a mutation of an active site residue, such as His223, Glu224, His227, Glu262, and/or Tyr366. The position numbering corresponds to the amino acid positions of SEQ ID NO: 62 and can be determined by aligning a polypeptide with SEQ ID NO: 62. As the presence of a methionine residue at position 1 of SEQ ID NO: 62 is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 62 includes a methionine, the position numbering will be as defined above (e.g. His223 will be His223 of SEQ ID NO: 62). Alternatively, where the methionine is absent from SEQ ID NO: 62 the amino acid residue numbering should be modified by −1 (e.g. His223 will be His222 of SEQ ID NO: 62). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present/absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.
In a particularly preferred embodiment, a polypeptide of the invention may comprise a modified BoNT/A or fragment thereof (preferably a BoNT/A HC domain or fragment thereof). The modified BoNT/A or fragment thereof may be one that comprises a modification at one or more amino acid residue(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. Such a modified BoNT/A or fragment thereof may demonstrate a reduction in, or absence of, side effects compared to the use of known BoNT/A. The increased tissue retention properties of the modified BoNT/A of the invention may 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 adverse effects), thus providing further advantages.
The modification may be a modification when compared to unmodified BoNT/A shown as SEQ ID NO: 62, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 62. As the presence of a methionine residue at position 1 of SEQ ID NO: 62 (as well as the SEQ ID NOs corresponding to modified BoNT/A polypeptides or fragments thereof described herein) is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 62 includes a methionine, the position numbering will be as defined above (e.g. ASN 886 will be ASN 886 of SEQ ID NO: 62). Alternatively, where the methionine is absent from SEQ ID NO: 2 the amino acid residue numbering should be modified by −1 (e.g. ASN 886 will be ASN 885 of SEQ ID NO: 62). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present/absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.
The amino acid residue(s) indicated for modification above are surface exposed amino acid residue(s).
A modified BoNT/A or fragment thereof may comprise a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277.
The term “one or more amino acid residue(s)” when used in the context of modified BoNT/A or fragment thereof preferably means at least 2, 3, 4, 5, 6 or 7 of the indicated amino acid residue(s). Thus, a modified BoNT/A may comprise at least 2, 3, 4, 5, 6 or 7 (preferably 7) modifications at the indicated amino acid residue(s). A modified BoNT/A or fragment thereof may comprise 1-30, 3-20, or 5-10 amino acid modifications. More preferably, the term “one or more amino acid residue(s)” when used in the context of modified BoNT/A or fragment thereof means all of the indicated amino acid residue(s).
Preferably, beyond the one or more amino acid modification(s) at the indicated amino acid residue(s), the modified BoNT/A or fragment thereof does not contain any further amino acid modifications when compared to SEQ ID NO: 62.
The modification may be selected from:
A modification as indicated above results in a modified BoNT/A or fragment thereof that has an increased positive surface charge and increased isoelectric point when compared to the corresponding unmodified BoNT/A or fragment thereof.
The isoelectric point (pl) 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 residues). 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 hydrophobicity. These properties may be influenced by the surrounding chemical environment, such as the temperature and pH. The overall chemical characteristics of a protein will depend on the sum of these various factors.
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 relevant 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 strength 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 overall (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 charge. This point is known as the isoelectric point (pl). The isoelectric point is a standard concept in protein biochemistry with which the skilled person would be familiar.
The isoelectric point (pl) is therefore defined as the pH value at which a protein displays a net charge of zero. An increase in pl means that a higher pH value is required for the protein to display a net charge of zero. Thus, an increase in pl represents an increase in the net positive charge of a protein at a given pH. Conversely, a decrease in pl means that a lower pH value is required for the protein to display a net charge of zero. Thus, a decrease in pl represents a decrease in the net positive charge of a protein at a given pH.
Methods of determining the pl of a protein are known in the art and would be familiar to a skilled person. By way of example, the pl of a protein can be calculated from the average pKa values of each amino acid present in the protein (“calculated pl”). Such calculations can be performed using computer programs known in the art, such as the Compute pl/MW Tool from ExPASy (https://web.expasy.org/compute_pi/), which is the preferred method for calculating pl in accordance with the present invention. Comparisons of pl values between different molecules should be made using the same calculation technique/program.
Where appropriate, the calculated pl of a protein can be confirmed experimentally using the technique of isoelectric focusing (“observed pl”). This technique uses electrophoresis to separate proteins according to their pl. Isoelectric focusing is typically performed using a gel that has an immobilised pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches the pH at which it has zero net charge, this point being the pl of the protein. Results provided by isoelectric focusing are typically relatively low-resolution in nature, and thus the present inventors believe that results provided by calculated pl (as described above) are more appropriate to use.
Throughout the present specification, “pl” means “calculated pl” unless otherwise stated.
The pl of a protein may be increased 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 protein. For example, an increase in pl may be provided by reducing the number of acidic residues, or by increasing the number of basic residues.
A modified BoNT/A or fragment thereof of the invention may have a pl value that is at least 0.2, 0.4, 0.5 or 1 pl units higher than that of an unmodified BoNT/A (e.g. SEQ ID NO: 62) or fragment thereof. Preferably, a modified BoNT/A or fragment thereof may have a pl of at least 6.6, e.g. at least 6.8.
The properties of the 20 standard amino acids are indicated in the table below:
The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).
At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.
The following amino acids are considered uncharged, polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.
The following amino acids are considered uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.
In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into the BoNT/A polypeptide sequence or fragment thereof, 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 sequence, thus reducing the total number of amino acid residues in said sequence.
Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT/A or fragment thereof. In an amino acid substitution, an amino acid residue that forms part of the BoNT/A polypeptide sequence or fragment thereof is replaced with a different amino acid residue. The replacement amino acid residue may be one of the 20 standard 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 non-standard amino acid, e.g. L-Ornithine, L-2-amino-3-guanidinopropionic acid, or D-isomers of Lysine, Arginine and Ornithine). Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using E. coli auxotrophic expression hosts.
In one embodiment, the substitution is selected from: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, 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 residue (i.e. aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine).
Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is substitution with lysine or arginine. Most preferably, the modification is substitution with lysine.
Preferably, a modified BoNT/A or fragment thereof for use in the invention comprises between 4 and 40 amino acid modifications located in the clostridial toxin HN domain. Said modified BoNT/A or fragment thereof preferably also has pl of at least 6.6. Said modified BoNT/A preferably comprises modifications of at least 4 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, and ASN 1052, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue. For example, said modified BoNT/A or fragment thereof may comprise modifications of at least 5 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue.
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 polypeptide (e.g. encoding unmodified BoNT/A or a fragment thereof). This can be achieved using standard molecular cloning techniques, for example by site-directed 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 enzyme, or by inserting/deleting parts of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, a modified gene sequence can be chemically synthesised.
In one aspect the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and/or wherein the polypeptide comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41.
In a related aspect, there is provided a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and/or wherein the polypeptide comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41.
In a further related aspect, there is provided use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and/or wherein the polypeptide comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41.
In one aspect the invention provides a polypeptide for use in treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and/or wherein the polypeptide comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41.
In a related aspect, there is provided a method for treating a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and/or wherein the polypeptide comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41.
In a further related aspect, there is provided use of a polypeptide in the manufacture of a medicament for treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and/or wherein the polypeptide comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41.
In one embodiment a polypeptide for use according to the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 42. Preferably, a polypeptide for use according to the invention comprises a polypeptide sequence shown as SEQ ID NO: 42.
In one embodiment a polypeptide for use according to the invention comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 41. Preferably, a polypeptide for use according to the invention comprises a polypeptide sequence that is encoded by a nucleotide sequence shown as SEQ ID NO: 41.
In one embodiment a polypeptide for use according to the invention (e.g. comprising SEQ ID NO: 42 or encoded by SEQ ID NO: 41) may be a portion of a polypeptide having at least 70% sequence identity to SEQ ID NO: 61 or 65. Thus, in one embodiment a polypeptide for use according to the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 61 or 65. Preferably, a polypeptide for use according to the invention may comprise (more preferably consist of) SEQ ID NO: 61 or 65. In one embodiment the polypeptide comprises a catalytically-inactive L-chain (e.g. as per SEQ ID NO: 65).
In one embodiment a polypeptide for use according to the invention (e.g. comprising SEQ ID NO: 42 or encoded by SEQ ID NO: 41) may be encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 60. Thus, in one embodiment a polypeptide for use according to the invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 60. Preferably, a polypeptide for use according to the invention may be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 60. In one embodiment the polypeptide comprises a catalytically-inactive L-chain.
SEQ ID NO: 42 is an example of a modified BoNT/A fragment and SEQ ID NOs: 61 and 65 are examples of modified BoNT/A polypeptides that are catalytically active and inactive, respectively. Such modified BoNT/A polypeptides and fragments are particularly preferred for use in the present invention. The polypeptides shown as SEQ ID NO: 42, 61 and 62 have a number of amino acid modifications (e.g. substitutions) when compared to wild-type BoNT/A, which increase the isoelectric point of the polypeptide. Without wishing to be bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and cell surface thus increasing retention at a site of administration and/or duration of action. Thus, it is envisaged that neuronal growth and/or repair properties of SEQ ID NO: 42, 61 and 65 will be improved compared to equivalent polypeptides lacking said modifications.
For the catalytically active modified BoNT/A polypeptides described above (e.g. SEQ ID NO: 61), one way in which these advantageous properties (which represent an increase in the therapeutic index) may be defined is in terms of the Safety Ratio of the modified BoNT/A. In this regard, undesired effects of a clostridial neurotoxin (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 (e.g. a mouse, where loss of bodyweight is detected within seven days of administration). Conversely, desired on-target effects of a clostridial neurotoxin 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 μl of clostridial neurotoxin, formulated in Gelatin Phosphate Buffer, into the mouse gastrocnemius/soleus complex, followed by assessment of Digital Abduction Score using the method of Aoki (Aoki K R, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are suspended briefly by the tail in order to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. Following clostridial neurotoxin injection, the varying degrees of digit abduction are scored on a five-point scale (0=normal to 4=maximal reduction in digit abduction and leg extension).
The Safety Ratio of a clostridial neurotoxin may then be expressed as the ratio between 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 required for a DAS score of 2. High Safety Ratio scores are therefore desired and indicate a toxin that is able to effectively paralyse a target muscle with little undesired off-target effects. A catalytically active modified BoNT/A of the present invention may have a Safety Ratio that is higher than the Safety Ratio of an equivalent unmodified (native) botulinum toxin (e.g. SEQ ID NO: 62).
Thus, in one embodiment, a catalytically active modified BoNT/A 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 required to produce a DAS score of 2].
In one embodiment, a catalytically active modified BoNT/A of the present invention has a Safety Ratio of at least 10. In one embodiment, a modified BoNT/A or fragment thereof of the present invention has a Safety Ratio of at least 15.
Polypeptides comprising at least 70% sequence identity to SEQ ID NO: 61 are described in WO 2015/004461 A1, which is incorporated herein by reference in its entirety.
In one embodiment a polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, 61 or 65 and/or comprising a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 or 60 comprises a substitution at one or more (preferably two or more, three or more, four or more, five or more or six or more, more preferably at all) of positions 930, 955, 991, 1026, 1052, 1229, and 886. The position numbering corresponds to the positions of SEQ ID NO: 62 and can be determined by aligning the polypeptide sequence with SEQ ID NO: 62 (unmodified/wild-type BoNT/A). As the presence of a methionine residue at position 1 of SEQ ID NO: 62 is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 62 includes a methionine, the position numbering will be as defined above (e.g. position 886 will be ASN 886 of SEQ ID NO: 62). Alternatively, where the methionine is absent from SEQ ID NO: 62 the amino acid residue numbering should be modified by −1 (e.g. position 886 will be ASN 885 of SEQ ID NO: 62). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present/absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.
Preferably, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, 61 or 65 and/or comprising a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 or 60 comprises lysine or arginine (more preferably lysine) at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, the polypeptide comprises lysine or arginine (more preferably lysine) at least two, three, four, five, six or all of positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the polypeptide comprises lysine or arginine (more preferably lysine) at all of positions 930, 955, 991, 1026, 1052, 1229, and 886.
The polypeptides of the invention promote neuronal growth and/or neuronal repair. Thus, said polypeptides find utility in treating neurological disorders. The term “neurological disorder” as used herein is a disorder that can be treated by promoting neuronal growth and/or repair in a subject.
Thus, in one aspect the invention provides a method for promoting neuronal growth and/or neuronal repair, the method comprising administering a polypeptide to a subject, the polypeptide comprising a clostridial neurotoxin light chain (L-chain) or fragment thereof; and/or a fragment of a clostridial neurotoxin heavy chain (H-chain). In another aspect, the invention provides a method for promoting neuronal growth and/or neuronal repair, the method comprising administering a polypeptide to a subject, the polypeptide comprising a catalytically inactive clostridial neurotoxin L-chain. In another aspect, there is provided a method for promoting neuronal growth or neuronal repair, the method comprising administering a polypeptide to a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and/or wherein the polypeptide comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41. In another aspect there is provided a method for promoting neuronal growth or neuronal repair, the method comprising administering a polypeptide to a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63.
The term “promotes neuronal growth and/or neuronal repair” may mean that the polypeptide of the invention initiates neuronal growth and/or neuronal repair, for example where neuronal growth and/or neuronal repair was not occurring. In other embodiments, the term “promotes neuronal growth and/or neuronal repair” may mean that the polypeptide of the invention increases the rate of neuronal growth and/or neuronal repair. Said increase may be an increase when compared to the rate of neuronal growth and/or neuronal repair in the absence of the polypeptide of the invention. In one embodiment neuronal growth and/or neuronal repair allows for the rebuilding of damaged neuronal circuits, thereby restoring activity and/or neuronal communication in a network or population of neurons. Thus, the term “neuronal repair” as used herein may encompass repair of a specific neuron as well as repair of a neuronal circuit.
The term “neuronal growth and/or neuronal repair” may also encompass neuronal plasticity. Thus, in one embodiment a polypeptide of the invention promotes neuronal plasticity. The term “neuronal plasticity” as used herein encompasses axonal sprouting, dendritic sprouting, neurogenesis (e.g. the production of new neurons), maturation, differentiation, and/or synaptic plasticity (e.g. including changes to synaptic strength, activity, anatomy, and/or connectivity). In one embodiment a polypeptide of the invention promotes the establishment of functional synapses (e.g. at or near to a site of injury).
Neuronal growth and/or repair may be increased by at least 10%, 20%, 30%, 40%, 50%, 60% or 70% (preferably at least 80%) in the presence of a polypeptide of the invention when compared to the neuronal growth and/or repair in the absence of the polypeptide of the invention or in the presence of an alternative polypeptide. In some embodiments neuronal growth and/or repair may be increased by at least 100%, 150% or 200% in the presence of a polypeptide of the invention when compared to the neuronal growth and/or repair in the absence of the polypeptide of the invention or in the presence of an alternative polypeptide.
In one embodiment, a polypeptide of the invention promotes neuronal growth. The term “neuronal growth” as used herein encompasses growth of any part of a neuron, including growth of axons and/or dendrites. A polypeptide of the invention may increase neurite length, neurite number (e.g. number of neurites per cell), and/or may increase the length and/or numbers of projections from a cell body or cell membrane of a neuron. Preferably, a polypeptide of the invention promotes axonal growth of a neuron, e.g. a neuron in a subject. In other words, preferably a polypeptide of the invention increases axonal growth, e.g. axonal sprouting. Said axonal growth may promote connections and/or chemical communication between neurons.
A neurological disorder treated by a polypeptide of the invention may be a neuronal injury, a neurodegenerative disorder, a sensory disorder or an autonomic disorder.
A neurological disorder may be a neuronal injury. In one embodiment, a neuronal injury may be nerve trauma, neuropathy (e.g. peripheral neuropathy), spinal cord injury, a nerve section, brain injury (e.g. traumatic brain injury), non-traumatic injury (e.g. stroke or spinal cord infarction), or injury to the brachial plexus, e.g. Erb's palsy or Klumpke's palsy.
In one embodiment the nerve trauma may result from scarring and/or from a bone fracture. In such instances of nerve trauma, nerve terminals are damaged. The polypeptide of the invention, advantageously, allows for repair of said nerve terminals or of distal nerve terminals allowing treatment of nerve trauma.
A neuronal injury may be paralysis, such as paralysis caused by spinal cord injury (e.g. caused by compression, constriction, and/or stretching). In one embodiment a spinal cord injury is paraplegia or tetraplegia.
A neurological disorder may be a sensory disorder. In one embodiment, a sensory disorder is sensory neuropathy, sensorimotor polyneuropathy, diabetic neuropathy, pain, Brown-Sequard syndrome, Charcot-Marie-Tooth disease, or Devic's syndrome. Preferably, a sensory disorder described herein is not pain. In other words, preferably a neurological disorder described herein is not pain.
A neurological disorder may be an autonomic disorder. In one embodiment, an autonomic disorder is autonomic neuropathy, multiple system atrophy, acute idiopathic polyneuropathy, dysautonomia, familial dysautonomia, diabetic autonomic failure, pure autonomic failure, temperature regulation disorders, hyperhidrosis, neurally mediated syncope (vasovagal, micturition, cough, swallow and other situational forms), erectile dysfunction, orthostatic hypotension, postural tachycardia syndrome (PoTS), or Guillain-Barre syndrome.
A neurological disorder may be a neurodegenerative disorder. In one embodiment, a neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, Parkinson's disease related disorders, motor neuron disease, peripheral neuropathy, motor neuropathy, prion disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, monomelic amyotrophy, Friedreich's ataxia, Hallervorden-Spatz disease, or frontotemporal lobar degeneration. Preferably, a neurodegenerative disorder is Parkinson's disease or motor neuron disease. Advantageously, the polypeptides of the invention are believed to find utility in the treatment of neurodegenerative disorders owing to their ability to promote neuronal growth (e.g. including neuronal plasticity) and/or neuronal repair, and further owing to their ability to rebuild damaged neuronal circuits, thereby restoring activity and/or neuronal communication in a network or population of neurons.
The polypeptides of the invention may be considered neurotrophic polypeptides in view of their ability to promote neuronal growth and/or neuronal repair. A neuron described herein may be one or more selected from: a motor neuron (including an autonomic neuron), a sensory neuron, a spinal interneuron, and a cerebral interneuron. Thus, in one embodiment a polypeptide of the invention promotes the growth and/or repair of a motor neuron, a sensory neuron, and/or an interneuron. Preferably, a polypeptide of the invention promotes the growth and/or repair of a motor neuron.
A “subject” as used herein may be a mammal, such as a human or other mammal. Preferably “subject” means a human subject.
The term “disorder” as used herein also encompasses a “disease”. In one embodiment the disorder is a disease.
The term “treat” or “treating” as used herein encompasses prophylactic treatment (e.g. to prevent onset of a disorder) as well as corrective treatment (treatment of a subject already suffering from a disorder). Preferably “treat” or “treating” as used herein means corrective treatment.
The term “treat” or “treating” as used herein refers to the disorder and/or a symptom thereof.
Therefore a polypeptide of the invention may be administered to a subject in a therapeutically effective amount or a prophylactically effective amount. Preferably a polypeptide of the invention is administered to a subject in a therapeutically effective amount.
A “therapeutically effective amount” is any amount of the polypeptide, which when administered alone or in combination to a subject for treating said disorder (or a symptom thereof) is sufficient to effect such treatment of the disorder, or symptom thereof.
A “prophylactically effective amount” is any amount of the polypeptide that, when administered alone or in combination to a subject inhibits or delays the onset or reoccurrence of a disorder (or a symptom thereof). In some embodiments, the prophylactically effective amount prevents the onset or reoccurrence of a disorder entirely. “Inhibiting” the onset means either lessening the likelihood of a disorder's onset (or symptom thereof), or preventing the onset entirely.
The polypeptides of the invention may be formulated in any suitable manner for administration to a subject, for example as part of a pharmaceutical composition. Thus, in one aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt. In some embodiments, the polypeptide of the invention may be in a single-chain form, while in other embodiments the polypeptide may be in a di-chain form, e.g. where the two chains are linked by a di-sulphide bridge. Preferably the polypeptide is in a di-chain form.
The polypeptides 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 a polypeptide that is to be delivered locally, the polypeptide may be formulated 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 a polypeptide enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.
Polypeptides of the invention may be administered to a subject 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 route of administration may be via laproscopic and/or localised injection. In one embodiment a polypeptide of the invention is administered at or near to a site of injury, preferably at a site of injury. For example, where an injury is a spinal cord injury, the polypeptide may be administered intrathecally or intraspinally (preferably intrathecally). In one embodiment the route of administration of a polypeptide of the invention may be perineural, intraneural, intraspinal, and/or intrathecal.
The dosage ranges for administration of the polypeptides of the present invention are those to produce the desired therapeutic and/or prophylactic effect. It will be appreciated that the dosage range required depends on the precise nature of the clostridial neurotoxin or composition, the route of administration, the nature of the formulation, the age of the subject, the nature, extent or severity of the subject's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
In one embodiment a dosage of the polypeptide is a flat dose. A flat dose may be in the range of 50 pg to 250 ug, preferably 100 pg to 100 ug. In one embodiment a flat dose may be at least 50 pg, 100 pg, 500 pg, 1 ng, 50 ng, 100 ng, 500 ng, 1 ug or 50 ug. Said dose may be a single flat dose.
Fluid dosage forms are typically prepared utilising the polypeptide and a pyrogen-free sterile vehicle. The clostridial neurotoxin, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. In preparing solutions the polypeptide can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or 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 an administration route described herein, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
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, or high-pressure aerosol impingement.
A polypeptide of the invention may be a clostridial neurotoxin or a fragment thereof, preferably a fragment thereof.
In one embodiment, a polypeptide of the invention may be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60. In one embodiment, a polypeptide of the invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60. Preferably, a polypeptide of the invention may be encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60.
In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65. In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65. Preferably, a polypeptide of the invention may comprise a polypeptide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
In one embodiment the present invention encompasses the use of full-length clostridial neurotoxins comprising a clostridial neurotoxin L-chain and a clostridial neurotoxin H-chain with the proviso that said clostridial neurotoxin L-chain is catalytically inactive.
The term “clostridial neurotoxin” embraces toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified clostridial neurotoxins or derivatives derived from any of the foregoing.
Botulinum 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 X all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralising anti-sera, with such classification by serotype correlating with percentage sequence identity at the amino acid level. BoNT proteins 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 prevent the release of their neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/C1, BoNT/A and BoNT/E cleave the synaptosomal-associated protein of 25 kDa (SNAP-25); and BoNT/C1 cleaves syntaxin. BoNT/X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.
Tetanus toxin is produced in a single serotype by C. tetani. C. butyricum produces BoNT/E, while C. baratii produces BoNT/F.
The term “clostridial neurotoxin” is also intended to embrace modified clostridial neurotoxins and derivatives thereof, including but not limited to those described below. A modified clostridial neurotoxin or derivative may contain one or more amino acids that has been modified as compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. By way of example, a modified clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify functional aspects of the toxin, for example biological activity or persistence. Thus, in one embodiment, the clostridial neurotoxin of the invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.
A modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified HC domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial neurotoxin. 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 protein receptor of the target nerve cell. Examples of such modified clostridial neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are hereby incorporated by reference in their entirety.
A modified clostridial neurotoxin 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 L-chain. Examples of such modified clostridial neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are hereby incorporated by reference in their entirety.
A modified clostridial neurotoxin may comprise one or more modifications that increases or decreases the biological activity and/or the biological persistence of the modified clostridial neurotoxin. For example, a modified clostridial neurotoxin may comprise a leucine- or tyrosine-based motif, wherein said motif increases or decreases the biological activity and/or the biological persistence of the modified clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxlL, 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 clostridial neurotoxins comprising leucine- and tyrosine-based motifs are described in WO 2002/08268, which is hereby incorporated by reference in its entirety.
As described above, a modified clostridial neurotoxin (or clostridial neurotoxin fragment) may be one that comprises one or more modifications that increases the isoelectric point of the clostridial neurotoxin when compared to an equivalent unmodified clostridial neurotoxin lacking said one or more modifications. Suitable modified clostridial neurotoxins are described above and in WO 2015/004461 A1 and WO 2016/110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 61 and 42 described herein.
The term “clostridial neurotoxin” is intended to embrace hybrid and chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof, and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment the hybrid clostridial neurotoxin may contain the entire light chain of a light chain from one clostridial neurotoxin subtype and the heavy chain from another clostridial neurotoxin subtype. In another embodiment, a chimeric clostridial neurotoxin may contain a portion (e.g. the binding domain) of the heavy chain of one clostridial neurotoxin subtype, with another portion of the heavy chain being from another clostridial neurotoxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain portions from different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of such clostridial neurotoxins to subjects who are immunologically resistant to a given clostridial neurotoxin subtype, to subjects who may have a lower than average concentration of receptors to a given clostridial neurotoxin heavy chain binding domain, or to subjects 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 neurotoxins are described in U.S. Pat. No. 8,071,110, which publication is hereby incorporated by reference in its entirety. Thus, in one embodiment, the clostridial neurotoxin (or fragment thereof) of the invention is a hybrid clostridial neurotoxin, or a chimeric clostridial neurotoxin.
In a particularly preferred embodiment, a polypeptide of the invention may be a chimeric clostridial neurotoxin comprising (preferably consisting of) a BoNT/A light-chain and translocation domain, and a BoNT/B receptor binding domain (HC domain) or a portion thereof. A suitable chimeric and/or hybrid clostridial neurotoxin may be one taught in WO 2017/191315 A1, which is incorporated herein by reference. Such preferred sequences include SEQ ID NOs: 44, 63, and 64.
The BoNT/A LHN domain may be covalently linked to the BoNT/B HC domain. Said chimeric BoNT/A is also referred to herein as “BoNT/AB” or a “BoNT/AB chimera”.
The C-terminal amino acid residue of the LHN domain may correspond to the first amino acid residue of the 310 helix separating the LHN and HC domains of BoNT/A, and the N-terminal amino acid residue of the HC domain may correspond to the second amino acid residue of the 310 helix separating the LHN and HC domains in BoNT/B.
Reference herein to the “first amino acid residue of the 310 helix separating the LHN and HC domains of BoNT/A” means the N-terminal residue of the 310 helix separating the LHN and HC domains.
Reference herein to the “second amino acid residue of the 310 helix separating the LHN and HC domains of BoNT/B” means the amino acid residue following the N-terminal residue of the 310 helix separating the LHN and HC domains.
A “310 helix” is a type of secondary structure found in proteins and polypeptides, along with α-helices, β-sheets and reverse turns. The amino acids in a 310 helix are arranged in a right-handed helical structure where each full turn is completed by three residues and ten atoms that separate the intramolecular hydrogen bond between them. Each amino acid corresponds to a 120° turn in the helix (i.e., the helix has three residues per turn), and a translation of 2.0 Å (=0.2 nm) along the helical axis, and has 10 atoms in the ring formed by making the hydrogen bond. Most importantly, the N—H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues earlier; this repeated i+3→i hydrogen bonding defines a 310 helix. A 310 helix is a standard concept in structural biology with which the skilled person is familiar.
This 310 helix corresponds to four residues which form the actual helix and two cap (or transitional) residues, one at each end of these four residues. The term “310 helix separating the LHN and HC domains” as used herein consists of those 6 residues.
Through carrying out structural analyses and sequence alignments, a 310 helix separating the LHN and HC domains was identified. This 310 helix is surrounded by an α-helix at its N-terminus (i.e. at the C-terminal part of the LHN domain) and by a 6-strand at its C-terminus (i.e. at the N-terminal part of the HC domain). The first (N-terminal) residue (cap or transitional residue) of the 310 helix also corresponds to the C-terminal residue of this α-helix.
The 310 helix separating the LHN and HC domains can be for example determined from publicly available crystal structures of botulinum neurotoxins, for example 3BTA (http://www.rcsb.org/pdb/explore/explore.do?structureld=3BTA) and 1 EPW (http://www.rcsb.org/pdb/explore/explore.do?structureld=1EPW) for botulinum neurotoxins A1 and B1 respectively.
In silico modelling and alignment tools which are publicly available can also be used to determine the location of the 310 helix separating the LHN and HC domains in other neurotoxins, for example the homology modelling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http://loopp.org), PHYRE (Protein Homology/analogY Recognition Engine, http://www.sbg.bio.ic.ac.uk/phyre2/) and Rosetta (https://www.rosettacommons.org/), the protein superposition server SuperPose (http://wishart.biology.ualberta.ca/superpose/), the alignment program Clustal Omega (http://www.clustal.org/omega/), and a number of other tools/services listed at the Internet Resources for Molecular and Cell Biologists (http://molbiol-tools.ca/). In particular that the region around the “HN/HCN” junction is structurally highly conserved which renders it an ideal region to superimpose different serotypes.
For example, the following methodology may be used to determine the sequence of this 310 helix in other neurotoxins:
Examples of LHN, HC and 310, helix domains determined by this method are presented below:
872NIINTS877
872NIVNTS877
872NIVNTS877
872NITNAS877
872NIINTS877
872NIINTS877
872NIINTS877
872NITNTS877
859EILNNI864
859EILNNI864
859EILNNI864
859EILNNI864
859DILNNI864
859EILNNI864
859EILNNI864
859EILNNI864
Using structural analysis and sequence alignments, it was found that the β-strand following the 310 helix separating the LHN and HC domains is a conserved structure in all botulinum and tetanus neurotoxins and starts at the 8th residue when starting from the first residue of the 310 helix separating the LHN and HC domains (e.g., at residue 879 for BoNT/A1).
A BoNT/AB chimera may comprise an LHN domain from BoNT/A covalently linked to a HC domain from BoNT/B,
A BoNT/AB chimera may comprise an LHN domain from BoNT/A covalently linked to a HC domain from BoNT/B,
The rationale of the design process of the BoNT/AB chimera was to try to ensure that the secondary structure was not compromised and thereby minimise any changes to the tertiary structure and to the function of each domain. Without wishing to be bound by theory, it is hypothesized that by not disrupting the four central amino acid residues of the 310 helix in the BoNT/AB chimera ensures an optimal conformation for the chimeric neurotoxin, thereby allowing for the chimeric neurotoxin to exert its functions to their full capacity.
The LHN domain from BoNT/A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 62, or a polypeptide sequence having at least 70% sequence identity thereto. The LHN domain from BoNT/A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 62, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. Preferably, the LHN domain from BoNT/A corresponds to amino acid residues 1 to 872 of SEQ ID NO: 62.
The HC domain from BoNT/B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 70% sequence identity thereto. The HC domain from BoNT/B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. Preferably, the HC domain from BoNT/B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 52.
Preferably, the BoNT/AB chimera comprises a BoNT/A LHN domain and a BoNT/B HC domain. More preferably, the LHN domain corresponds to amino acid residues 1 to 872 of BoNT/A (SEQ ID NO: 62) and the HC domain corresponds to amino acid residues 860 to 1291 of BoNT/B (SEQ ID NO: 52).
Preferably, a BoNT/B HC domain further comprises at least one amino acid residue substitution, addition or deletion in the HCC subdomain which has the effect of increasing the binding affinity of BoNT/B neurotoxin for human Syt II as compared to the natural BoNT/B sequence. Suitable amino acid residue substitution, addition or deletion in the BoNT/B HCC subdomain have been disclosed in WO 2013/180799 and in WO 2016/154534 (both herein incorporated by reference).
Suitable amino acid residue substitution, addition or deletion in the BoNT/B HCC subdomain include substitution mutations selected from the group consisting of: V1118M; Y1183M; E1191M; E11911; E1191Q; E1191T; 51199Y; 51199F; 51199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, 51199E, 51199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.
Suitable amino acid residue substitution, addition or deletion in the BoNT/B HCC subdomain further include combinations of two substitution mutations selected from the group consisting of: E1191M and 51199L, E1191M and 51199Y, E1191M and 51199F, E1191Q and 51199L, E1191Q and 51199Y, E1191Q and 51199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and 51199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and 51199Y, or E1191V and W1178Q.
Suitable amino acid residue substitution, addition or deletion in the BoNT/B HCC subdomain also include a combination of three substitution mutations which are E1191M, S1199W and W1178Q.
Preferably, the suitable amino acid residue substitution, addition or deletion in the BoNT/B HCC subdomain includes a combination of two substitution mutations which are E1191M and 51199Y.
The modification may be a modification when compared to unmodified BoNT/B shown as SEQ ID NO: 52, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 52. As the presence of a methionine residue at position 1 of SEQ ID NO: 52 is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 52 includes a methionine, the position numbering will be as defined above (e.g. E1191 will be E1191 of SEQ ID NO: 52). Alternatively, where the methionine is absent from SEQ ID NO: 52 the amino acid residue numbering should be modified by −1 (e.g. E1191 will be E1190 of SEQ ID NO: 52). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present/absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.
Thus, in one aspect, the invention provides a polypeptide for use in promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.
In a related aspect, there is provided a method for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.
In a further related aspect, there is provided use of a polypeptide in the manufacture of a medicament for promoting neuronal growth or neuronal repair to treat a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.
In one aspect the invention provides a polypeptide for use in treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.
In a related aspect, there is provided a method for treating a neurological disorder in a subject, the method comprising administering a polypeptide to the subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.
In a further related aspect, there is provided use of a polypeptide in the manufacture of a medicament for treating a neurological disorder in a subject, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64.
In one embodiment a polypeptide for use according to the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 63 or 64. Preferably, a polypeptide for use according to the invention comprises (more preferably consists of) a polypeptide sequence shown as SEQ ID NO: 63 or 64.
Preferably, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 comprises a catalytically-inactive L-chain, such as SEQ ID NO: 64.
A chimeric and/or hybrid clostridial neurotoxin for use in the present invention may comprise a portion of a BoNT/A polypeptide and a portion of a BoNT/B polypeptide, an example of which includes the polypeptide described herein as SEQ ID NO: 44.
Suitable chimeric clostridial neurotoxins may include BoNT/FA. Indeed, in a particularly preferred embodiment, a polypeptide of the invention may comprise BoNT/FA or a fragment thereof. Catalytically inactive forms of BoNT/FA are described herein as SEQ ID NO: 26 and 34. Suitable fragments of BoNT/FA are also described herein as SEQ ID NOs: 28, 30, and 32.
The term “clostridial neurotoxin” may also embrace newly discovered botulinum neurotoxin protein family members expressed by non-clostridial microorganisms, such as the Enterococcus encoded toxin which has closest sequence identity to BoNT/X, the Weissella oryzae encoded toxin called BoNT/Wo (NCBI Ref Seq: WP_027699549.1), which cleaves VAMP2 at W89-W90, the Enterococcus faecium encoded toxin (GenBank: 0T022244.1), which cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero encoded toxin (NCBI Ref.Seq: WP_034687872.1).
The polypeptide of the present invention may lack a functional HC domain of a clostridial neurotoxin and also lack any functionally equivalent exogenous ligand Targeting Moiety (TM).
Thus, in a particularly preferred embodiment, a clostridial neurotoxin of the invention is not a re-targeted clostridial neurotoxin. In a re-targeted clostridial neurotoxin, the clostridial neurotoxin is modified to include an exogenous 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 neurotoxin (e.g. the HC domain, or the HCC domain) may be removed. Re-targeting technology is described, for example, in: EP-B-0689459; WO 1994/021300; EP-B-0939818; U.S. Pat. Nos. 6,461,617; 7,192,596; WO 1998/007864; EP-B-0826051; U.S. Pat. Nos. 5,989,545; 6,395,513; 6,962,703; WO 1996/033273; EP-B-0996468; U.S. Pat. No. 7,052,702; WO 1999/017806; EP-B-1107794; U.S. Pat. No. 6,632,440; WO 2000/010598; WO 2001/21213; WO 2006/059093; WO 2000/62814; WO 2000/04926; WO 1993/15766; WO 2000/61192; and WO 1999/58571; all of which are hereby incorporated by reference in their entirety.
As discussed above, (full-length) clostridial neurotoxins 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 targeting component (receptor binding domain or HC domain) and an N-terminal translocation component (HN domain).
A clostridial neurotoxin may be selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, and TeNT (tetanus neurotoxin). Preferably, a clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, and BoNT/X.
In one embodiment the clostridial neurotoxin may be BoNT/A. A reference BoNT/A sequence is shown as SEQ ID NO: 51. In another embodiment the clostridial neurotoxin may be BoNT/B. A reference BoNT/B sequence is shown as SEQ ID NO: 52. In another embodiment the clostridial neurotoxin may be BoNT/C. A reference BoNT/C sequence is shown as SEQ ID NO: 53. In another embodiment the clostridial neurotoxin may be BoNT/D. A reference BoNT/D sequence is shown as SEQ ID NO: 54. In another embodiment the clostridial neurotoxin may be BoNT/E. A reference BoNT/E sequence is shown as SEQ ID NO: 55. In another embodiment the clostridial neurotoxin may be BoNT/F. A reference BoNT/F sequence is shown as SEQ ID NO: 56. In another embodiment the clostridial neurotoxin may be BoNT/G. A reference BoNT/G sequence is shown as SEQ ID NO: 57. In another embodiment the clostridial neurotoxin may be TeNT. A reference TeNT sequence is shown as SEQ ID NO: 58. In another embodiment the clostridial neurotoxin may be BoNT/X. A reference BoNT/X sequence is shown as SEQ ID NO: 59.
In one embodiment a polypeptide of the invention comprises a fragment of a BoNT/A or a fragment of a BoNT/F. In another embodiment, the polypeptide of the invention comprises a catalytically inactive L-chain of BoNT/A or BoNT/F.
In embodiments where a polypeptide described herein has a tag for purification (e.g. a His-tag) and/or a linker, said tag and/or linker are optional.
Suitable full-length clostridial neurotoxins are described herein.
In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65 with the proviso that a clostridial neurotoxin L-chain of said polypeptide is catalytically inactive. In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65 with the proviso that a clostridial neurotoxin L-chain of said polypeptide is catalytically inactive. Preferably, a polypeptide of the invention may comprise a polypeptide sequence comprising any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65 with the proviso that a clostridial neurotoxin L-chain of said polypeptide is catalytically inactive.
In one embodiment a polypeptide of the invention may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60 with the proviso that the clostridial neurotoxin L-chain of said polypeptide is catalytically inactive. In one embodiment a polypeptide of the invention is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60 with the proviso that the clostridial neurotoxin L-chain of said polypeptide is catalytically inactive. Preferably, a polypeptide of the invention is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60 with the proviso that the clostridial neurotoxin L-chain of said polypeptide is catalytically inactive.
In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65 with the proviso that the clostridial neurotoxin L-chain of said polypeptide is catalytically inactive. In one embodiment a polypeptide of the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65 with the proviso that the clostridial neurotoxin L-chain of said polypeptide is catalytically inactive. Preferably, a polypeptide of the invention comprises any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65 with the proviso that the clostridial neurotoxin L-chain of said polypeptide is catalytically inactive.
In one embodiment a polypeptide of the invention is a full-length clostridial neurotoxin selected from BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, and TeNT.
In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63. In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63. In one embodiment a polypeptide of the invention may comprise a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63. Preferably, a polypeptide of the invention may comprise (more preferably consist of) a polypeptide sequence comprising any one of SEQ ID NOs: 52-59, 61 or 63.
In a particularly preferred embodiment a polypeptide of the invention is not a full-length catalytically active clostridial neurotoxin, e.g. is not full-length catalytically active BoNT/A.
The polypeptide of the present invention may comprise (or consist of) a fragment of a clostridial neurotoxin, e.g. a fragment of any full-length clostridial neurotoxin described herein.
In one embodiment a polypeptide of the invention may comprise a fragment of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65. In one embodiment a polypeptide of the invention may comprise a fragment of a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65. Preferably, a polypeptide of the invention may comprise a fragment of a polypeptide sequence comprising any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
In one embodiment a polypeptide of the invention comprises (or consists of) a clostridial neurotoxin L-chain or fragment thereof. A fragment of a clostridial neurotoxin L-chain may have 400, 350, 300, 250, 200, 150, 100 or 50 amino acid residues of a clostridial neurotoxin L-chain. In one embodiment, a fragment of a clostridial neurotoxin L-chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin L-chain. For example, a fragment of a clostridial neurotoxin L-chain may have 20-400, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin L-chain.
Examples of L-chain reference sequences include:
For recently-identified BoNT/X, the L-chain has been reported as corresponding to amino acids 1-439 thereof, with the L-chain boundary potentially varying by approximately 25 amino acids (e.g. 1-414 or 1-464).
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:
Suitable clostridial neurotoxin L-chains are described herein.
A clostridial neurotoxin L-chain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 6, 24, 32 or 40 or a fragment thereof. In one embodiment a clostridial neurotoxin L-chain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 6, 24, 32 or 40 or a fragment thereof. Preferably, a clostridial neurotoxin L-chain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 6, 24, 32 or 40 or a fragment thereof.
A clostridial neurotoxin L-chain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 5, 23, 31 or 39 or a fragment thereof. In one embodiment a clostridial neurotoxin L-chain is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 5, 23, 31 or 39 or a fragment thereof. Preferably, a clostridial neurotoxin L-chain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 5, 23, 31 or 39 or a fragment thereof.
In one embodiment a polypeptide of the invention comprises (or consists of) a fragment of a clostridial neurotoxin H-chain. A fragment of a clostridial neurotoxin H-chain may have 800, 700, 600, 500, 400, 350, 300, 250, 200, 150, 100 or 50 amino acid residues of a clostridial neurotoxin H-chain. In one embodiment, a fragment of a clostridial neurotoxin H-chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin H-chain. For example, a fragment of a clostridial neurotoxin H-chain may have 20-800, 30-600, 40-400, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin H-chain.
A clostridial neurotoxin H-chain comprises two structural/functional domains: the translocation domain (HN) and receptor binding domain (HC).
In one embodiment a polypeptide of the invention comprises (or consists of) a clostridial neurotoxin translocation domain or a fragment thereof. A fragment of a clostridial neurotoxin translocation domain may have 400, 350, 300, 250, 200, 150, 100 or 50 amino acid residues of a clostridial neurotoxin translocation domain. In one embodiment, a fragment of a clostridial neurotoxin translocation domain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin translocation domain. For example, a fragment of a clostridial neurotoxin translocation domain may have 20-400, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin translocation domain.
The translocation domain is a fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H-chain, or the domain corresponding to that fragment in the intact H-chain. In one embodiment the HC function of the H-chain may be removed by deletion of the HC amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the HC function may be inactivated by chemical or biological treatment. Thus, in some embodiments the H-chain may be incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.
Examples of suitable (reference) Translocation Domains include:
The above-identified reference sequence 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 thereto) cites slightly different clostridial sequences:
In the context of the present invention, a variety of clostridial neurotoxin HN regions comprising a translocation domain can be useful in aspects of the present invention. in one embodiment these active fragments can facilitate the release of a non-cytotoxic protease (e.g. a clostridial L-chain) from intracellular vesicles into the cytoplasm of the target cell and thus participate in executing the overall cellular mechanism whereby a clostridial neurotoxin proteolytically cleaves a substrate. The HN regions from the heavy chains of clostridial neurotoxins are approximately 410-430 amino acids in length and comprise a translocation domain. Research has shown that the entire length of a HN region from a clostridial neurotoxin heavy chain is not necessary for the translocating activity of the translocation domain. Thus, aspects of this embodiment can include clostridial neurotoxin HN regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids. Other aspects of this embodiment can include clostridial neurotoxin HN regions comprising a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.
For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.
The term HN embraces naturally-occurring neurotoxin HN portions, and modified HN portions having amino acid sequences that do not occur in nature and/or synthetic amino acid residues. In one embodiment said modified HN portions still demonstrate the above-mentioned translocation function.
In a preferred embodiment a polypeptide of the invention comprises (or consists of) a clostridial neurotoxin receptor binding domain (HC) or a fragment thereof. A fragment of a clostridial neurotoxin receptor binding domain (HC) may have 350, 300, 250, 200, 150, 00 or 50 amino acid residues of a clostridial neurotoxin receptor binding domain (HC). In one embodiment, a fragment of a clostridial neurotoxin receptor binding domain (HC) has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin receptor binding domain (HC). For example, a fragment of a clostridial neurotoxin receptor binding domain (HC) may have 20-350, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin receptor binding domain (HC).
Examples of clostridial neurotoxin receptor binding domain (HC) reference sequences include:
For recently-identified BoNT/X, the HC domain has been reported as corresponding to amino acids 893-1306 thereof, with the domain boundary potentially varying by approximately 25 amino acids (e.g. 868-1306 or 918-1306).
A clostridial neurotoxin H-chain may further comprise a translocation facilitating domain. Said domain facilitates delivery of the L-chain into the cytosol of the target cell and are described, for example, in WO 08/008803 and WO 08/008805, each of which is herein incorporated by reference thereto.
By way of example, a translocation facilitating domain may comprise a clostridial neurotoxin HCN domain or a fragment or variant thereof. In more detail, a clostridial neurotoxin HCN translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, a clostridial neurotoxin HCN translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include:
The above sequence positions may vary a little according to serotype/sub-type, and further examples of suitable (reference) clostridial neurotoxin HCN domains include:
Suitable clostridial neurotoxin HC domains are described herein.
A clostridial neurotoxin HC domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48 or 50 or a fragment thereof. In one embodiment a clostridial neurotoxin HC domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48 or 50 or a fragment thereof. Preferably, a clostridial neurotoxin HC domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48 or 50 or a fragment thereof.
A clostridial neurotoxin HC domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof. In one embodiment a clostridial neurotoxin HC domain is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof. Preferably, a clostridial neurotoxin HC domain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof.
In one embodiment a clostridial neurotoxin HC domain for use in the invention is a variant BoNT/A HC domain. Said variant BoNT/A HC domain may comprise a modification of one or more amino acids residues selected from Y1117, F1252, H1253, and L1278. For example, a variant BoNT/A HC domain may comprise one or more (preferably two or more) of the following modifications Y1117V, F1252Y, H1253K, and L1278F or L1278H.
In one embodiment a variant BoNT/A HC domain comprises the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278F; or Y1117V, F1252Y, H1253K, and L1278H.
Preferably, a variant BoNT/A HC domain comprises the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278H.
The modification may be a modification when compared to unmodified BoNT/A shown as SEQ ID NO: 62, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 62. As the presence of a methionine residue at position 1 of SEQ ID NO: 62 is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering. For example, where SEQ ID NO: 62 includes a methionine, the position numbering will be as defined above (e.g. Y1117 will align against Y1117 of SEQ ID NO: 62). Alternatively, where the methionine is absent from SEQ ID NO: 62 the amino acid residue numbering should be modified by −1 (e.g. Y1117 will align against Y1116 of SEQ ID NO: 52). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present/absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.
A variant BoNT/A HC domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. Preferably, a variant BoNT/A HC domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof.
A variant BoNT/A HC domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 46 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. Preferably, a variant BoNT/A HC domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 46 or 50 or a fragment thereof.
A variant BoNT/A HC domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain be one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain be one encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. Preferably, a variant BoNT/A HC domain be one encoded by any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof.
A variant BoNT/A HC domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 45 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain be one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 45 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. In one embodiment a variant BoNT/A HC domain be one encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above. Preferably, a variant BoNT/A HC domain be one encoded by any one of SEQ ID NOs: 45 or 49 or a fragment thereof.
Any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain may be combined with non-clostridial translocation domain peptide or with clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin HCN translocation facilitating domain may be combined with a non-clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin HCN facilitating domain may be combined with a clostridial translocation domain peptide, examples of which include:
In some embodiments the clostridial neurotoxins of the present invention may lack a functional HC domain of a clostridial neurotoxin. In one embodiment, the clostridial neurotoxins preferably lack the last 50 C-terminal amino acids of a clostridial neurotoxin holotoxin. In another embodiment, the clostridial neurotoxins preferably lack the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of a clostridial neurotoxin holotoxin. Alternatively, the HC binding activity may be negated/reduced by mutagenesis—by way of example, referring to BoNT/A for convenience, modification of one or two amino acid residue mutations (W1266 to L and Y1267 to F) in the ganglioside binding pocket causes the HC region to lose its receptor binding function. Analogous mutations may be made to non-serotype A clostridial peptide components, e.g. a construct based on botulinum B with mutations (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F). Other mutations to the active site achieve the same ablation of HC receptor binding activity, e.g. Y1267S in botulinum type A toxin and the corresponding highly conserved residue in the other clostridial neurotoxins. Details of this and other mutations are described in Rummel et al (2004) (Molecular Microbiol. 51:631-634), which is hereby incorporated by reference thereto.
The HC peptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25 kDa each, namely the N-terminal region (commonly referred to as the HCN peptide or domain) and the C-terminal region (commonly referred to as the HCC peptide or domain). This fact is confirmed by the following publications, each of which is herein incorporated in its entirety by reference thereto: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268:15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Moreover, it has been well documented that the C-terminal region (Hcc), which constitutes the C-terminal 160-200 amino acid residues, is responsible for binding of a clostridial neurotoxin to its natural cell receptors, namely to nerve terminals at the neuromuscular junction—this fact is also confirmed by the above publications. Thus, reference throughout this specification to a clostridial heavy-chain lacking a functional heavy chain HC peptide (or domain) such that the heavy-chain is incapable of binding to cell surface receptors to which a native clostridial neurotoxin binds means that the clostridial heavy-chain simply lacks a functional HCC peptide. In other words, the HCC peptide region may be either partially or wholly deleted, or otherwise modified (e.g. through conventional chemical or proteolytic treatment) to reduce its native binding ability for nerve terminals at the neuromuscular junction.
Thus, in one embodiment, a clostridial neurotoxin HN peptide of the present invention lacks part of a C-terminal peptide portion (Hcc) of a clostridial neurotoxin and thus lacks the HC binding function of native clostridial neurotoxin. By way of example, in one embodiment, the C-terminally extended clostridial HN peptide lacks the C-terminal 40 amino acid residues, or the C-terminal 60 amino acid residues, or the C-terminal 80 amino acid residues, or the C-terminal 100 amino acid residues, or the C-terminal 120 amino acid residues, or the C-terminal 140 amino acid residues, or the C-terminal 150 amino acid residues, or the C-terminal 160 amino acid residues of a clostridial neurotoxin heavy-chain. In another embodiment, the clostridial HN peptide of the present invention lacks the entire C-terminal peptide portion (HCC) of a clostridial neurotoxin and thus lacks the HC binding function of native clostridial neurotoxin. By way of example, in one embodiment, the clostridial HN peptide lacks the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of a clostridial neurotoxin heavy-chain. By way of further example, the clostridial HN peptide of the present invention lacks a clostridial HCC reference sequence selected from the group consisting of:
The above-identified reference sequences should be considered a guide as slight variations may occur according to sub-serotypes.
In a preferred embodiment a polypeptide of the invention comprises (or consists of) a clostridial neurotoxin L-chain or fragment thereof and a fragment of a clostridial neurotoxin H-chain. For example, a polypeptide may comprise (or consist of) a clostridial neurotoxin L-chain or fragment thereof and a clostridial neurotoxin translocation domain (HN). Preferably, the polypeptide does not further comprise a clostridial neurotoxin receptor binding domain (HC) or at least the C-terminal portion of a clostridial neurotoxin receptor binding domain (Hcc). Thus, in one embodiment a polypeptide of the present invention lacks a C-terminal portion of a clostridial neurotoxin receptor binding domain (Hcc). Advantageously, such polypeptides lack the endogenous clostridial neurotoxin receptor binding capabilities and thus exhibit fewer off-target effects in a subject administered said polypeptide.
In one embodiment a polypeptide of the invention consists essentially of a clostridial neurotoxin L-chain or fragment thereof and/or a fragment of a clostridial neurotoxin H-chain. The term “consists essentially of” as used in this context means that the polypeptide does not further comprise one or more amino acid residues that confer additional functionality to the polypeptide, e.g. when administered to a subject. In other words, a polypeptide that “consists essentially of” a clostridial neurotoxin L-chain or fragment thereof and/or a fragment of a clostridial neurotoxin H-chain may further comprise one or more amino acid residues (to those of the clostridial neurotoxin L-chain or fragment thereof and/or fragment of a clostridial neurotoxin H-chain) but said one or more further amino acid residues do not confer additional functionality to the polypeptide, e.g. when administered to a subject. Additional functionality may include enzymatic activity, binding activity and/or any physiological activity whatsoever.
In one embodiment a polypeptide may comprise non-clostridial neurotoxin sequences in addition to any clostridial neurotoxin sequences. The non-clostridial neurotoxin sequences preferably do not disrupt the ability of a polypeptide of the invention to promote neuronal growth or neuronal repair. Preferably, the non-clostridial neurotoxin sequence is not one having catalytic activity, e.g. enzymatic activity. Preferably, the non-clostridial sequence is not one that binds to a cellular receptor. In other words, it is most preferred that the non-clostridial sequence is not a ligand for a cellular receptor. A cellular receptor may be a proteinaceous cellular receptor, such as an integral membrane protein. Examples of cellular receptors can be found in the IUPHAR Guide to Pharmacology Database, version 2019.4, available at https://www.guidetopharmacology.org/download.jsp#db_reports. Non-clostridial neurotoxin sequences may include tags to aid in purification, such as His-tags. It is preferred that any clostridial neurotoxin sequences comprised in said polypeptide consist of a clostridial neurotoxin L-chain or fragment thereof and/or a fragment of a clostridial neurotoxin H-chain. In one embodiment, the clostridial neurotoxin sequence comprised in said polypeptide may consist of a clostridial neurotoxin L-chain. In one embodiment, the clostridial neurotoxin sequence comprised in said polypeptide may consist of a clostridial neurotoxin translocation domain. In one embodiment, the clostridial neurotoxin sequence comprised in said polypeptide may consist of a clostridial neurotoxin receptor binding domain. In one embodiment, the clostridial neurotoxin sequence comprised in said polypeptide may consist of a clostridial neurotoxin L-chain and a clostridial neurotoxin translocation domain.
Suitable polypeptides comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain are described herein.
A clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4, 20, 28 or 36 or a fragment thereof. In one a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 4, 20, 28 or 36 or a fragment thereof. Preferably, a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 4, 20, 28 or 36 or a fragment thereof.
A clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3, 19, 27 or 35 or a fragment thereof. In one embodiment a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 3, 19, 27 or 35 or a fragment thereof. Preferably, a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 3, 19, 27 or 35 or a fragment thereof.
The polypeptides of the present invention may be free from the complexing proteins that are present in a naturally occurring clostridial neurotoxin complex.
The polypeptides of the present invention can be produced using recombinant nucleic acid technologies. Thus, in one embodiment, a polypeptide (as described above) is a recombinant polypeptide.
In one embodiment a nucleic acid (for example, a DNA) comprising a nucleic acid sequence encoding a polypeptide is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator.
In a preferred embodiment, the vector has a promoter selected from:
In another preferred embodiment, the vector has a promoter selected from:
The nucleic acid molecules 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 preferably designed in silico, 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.
The terms “nucleotide sequence” and “nucleic acid” are used synonymously herein. Preferably the nucleotide sequence is a DNA sequence.
A polypeptide of the invention (and especially any clostridial neurotoxin portion thereof) may be present as a single-chain or as a di-chain.
The invention provides a method of producing a single-chain polypeptide having a light chain and a heavy chain, the method comprising expressing a nucleic acid described herein in an expression host, lysing the host cell to provide a host cell homogenate containing the single-chain polypeptide, and isolating the single-chain polypeptide. In one aspect, the present invention provides a method of activating a polypeptide described herein, the method comprising contacting the polypeptide with a protease that hydrolyses a peptide bond in the activation loop of the polypeptide, thereby converting the (single-chain) polypeptide into a corresponding di-chain polypeptide (e.g. wherein the light chain and heavy chain are joined together by a disulphide bond).
The present invention therefore provides a di-chain polypeptide obtainable by a method of the invention.
Embodiments related to the various therapeutic uses of the invention are intended to be applied equally to methods of treatment, polypeptides of the invention, and vice versa.
Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M—A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).
Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).
The “percent sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. 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. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons 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.
The percent identity is then calculated as:
Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
Basic: arginine
Acidic: glutamic acid
Polar: glutamine
Hydrophobic: leucine
Aromatic: phenylalanine
Small: glycine
In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-am inoisobutyric acid, isovaline and α-methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, 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 translation of plasm ids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence 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. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.
This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.
Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein”, as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms “protein” and “polypeptide” are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a clostridial neurotoxin” includes a plurality of such candidate agents and reference to “the clostridial neurotoxin” includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.
Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.
Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional.
SEQ ID NO: 1—Nucleotide Sequence of Recombinant Catalytically Inactive BoNT/A (rBoNT/A(0))
SEQ ID NO: 2—Polypeptide Sequence of rBoNT/A(0)
SEQ ID NO: 3—Nucleotide Sequence of rLHN/A (light-chain plus translocation domain only).
SEQ ID NO: 4—Polypeptide Sequence of rLHN/A
SEQ ID NO: 5—Nucleotide Sequence of rL/A (light-chain only)
SEQ ID NO: 6—Polypeptide Sequence of rL/A
SEQ ID NO: 7—Nucleotide Sequence of rHC/A
SEQ ID NO: 8—Polypeptide Sequence of rHC/A
SEQ ID NO: 9—Nucleotide Sequence of rBoNT/B(0)
SEQ ID NO: 10—Polypeptide Sequence of rBoNT/B(0)
SEQ ID NO: 11—Nucleotide Sequence of rBoNT/C(0)
SEQ ID NO: 12—Polypeptide Sequence of rBoNT/C(0)
SEQ ID NO: 13—Nucleotide Sequence of rBoNT/E(0)
SEQ ID NO: 14—Polypeptide Sequence of rBoNT/E(0)
SEQ ID NO: 15—Nucleotide Sequence of rBoNT/F(0)
SEQ ID NO: 16—Polypeptide Sequence of rBoNT/F(0)
SEQ ID NO: 17—Nucleotide Sequence of rBoNT/A(0) (His-tagged)
SEQ ID NO: 18—Polypeptide Sequence of rBoNT/A(0) (His-tagged)
SEQ ID NO: 19—Nucleotide Sequence of rLHN/A (His-tagged)
SEQ ID NO: 20—Polypeptide Sequence of rLHN/A (His-tagged)
SEQ ID NO: 21—Nucleotide Sequence of rHC/A (His-tagged)
SEQ ID NO: 22—Polypeptide Sequence of rHC/A (His-tagged)
SEQ ID NO: 23—Nucleotide Sequence of rLC/A (His-tagged)
SEQ ID NO: 24—Polypeptide Sequence of rLC/A (His-tagged)
SEQ ID NO: 25—Nucleotide Sequence of rBoNT/FA(0) (His-tagged)
SEQ ID NO: 26—Polypeptide Sequence of rBoNT/FA(0) (His-tagged)
SEQ ID NO: 27—Nucleotide Sequence of rLHN/FA (His-tagged)
SEQ ID NO: 28—Polypeptide Sequence of rLHN/FA (His-tagged)
SEQ ID NO: 29—Nucleotide Sequence of rHC/FA (His-tagged)
SEQ ID NO: 30—Polypeptide Sequence of rHC/FA (His-tagged)
SEQ ID NO: 31—Nucleotide Sequence of rLC/FA (His-tagged)
SEQ ID NO: 32—Polypeptide Sequence of rLC/FA (His-tagged)
SEQ ID NO: 33—Nucleotide Sequence of rBoNT/F(0) (His-tagged)
SEQ ID NO: 34—Polypeptide Sequence of rBoNT/F(0) (His-tagged)
SEQ ID NO: 35—Nucleotide Sequence of rLHN/F (His-tagged)
SEQ ID NO: 36—Polypeptide Sequence of rLHN/F (His-tagged)
SEQ ID NO: 37—Nucleotide Sequence of rHC/F (His-tagged)
SEQ ID NO: 38—Polypeptide Sequence of rHC/F (His-tagged)
SEQ ID NO: 39—Nucleotide Sequence of rLC/F (His-tagged)
SEQ ID NO: 40—Polypeptide Sequence of rLC/F (His-tagged)
SEQ ID NO: 41—Nucleotide Sequence of Cationic rHC/A (His-tagged)
SEQ ID NO: 42—Polypeptide Sequence of Cationic rHC/A (His-tagged)
SEQ ID NO: 43—Nucleotide Sequence of rHC/AB (His-tagged)
SEQ ID NO: 44—Polypeptide Sequence of rHC/AB (His-tagged)
SEQ ID NO: 45—Nucleotide Sequence of rHC/A Variant Y1117V H1253K (His-tagged)
SEQ ID NO: 46—Polypeptide Sequence of rHC/A Variant Y1117V H1253K (His-tagged)
SEQ ID NO: 47—Nucleotide Sequence of rHC/A Variant Y1117V F1252Y H1253K L1278F (His-tagged)
SEQ ID NO: 48—Polypeptide Sequence of rHC/A Variant Y1117V F1252Y H1253K L1278F (His-tagged)
SEQ ID NO: 49—Nucleotide Sequence of rHC/A Variant Y1117V F1252Y H1253K L1278H (His-tagged)
SEQ ID NO: 50—Polypeptide Sequence of rHC/A Variant Y1117V F1252Y H1253K L1278H (His-tagged)
SEQ ID NO: 51—Polypeptide Sequence of BoNT/A—UniProt P10845
SEQ ID NO: 52—Polypeptide Sequence of BoNT/B—UniProt P10844
SEQ ID NO: 53—Polypeptide Sequence of BoNT/C—UniProt P18640
SEQ ID NO: 54—Polypeptide Sequence of BoNT/D—UniProt P19321
SEQ ID NO: 55—Polypeptide Sequence of BoNT/E—UniProt Q00496
SEQ ID NO: 56—Polypeptide Sequence of BoNT/F—UniProt A7GBG3
SEQ ID NO: 57—Polypeptide Sequence of BoNT/G—UniProt Q60393
SEQ ID NO: 58—Polypeptide Sequence of TeNT— UniProt P04958
SEQ ID NO: 59—Polypeptide Sequence of BoNT/X
SEQ ID NO: 60—Nucleotide Sequence of mrBoNT/A
SEQ ID NO: 61—Polypeptide Sequence of mrBoNT/A
SEQ ID NO: 62—Polypeptide Sequence of Unmodified BoNT/A1
SEQ ID NO: 63—Polypeptide Sequence of mrBoNT/AB
SEQ ID NO: 64—Polypeptide Sequence of mrBoNT/AB(0)
SEQ ID NO: 65—Polypeptide Sequence of mrBoNT/A(0)
Five catalytically inactive (i.e. endopeptidase inactive) botulinum neurotoxin (BoNT) serotypes were recombinantly expressed in E. coli, namely corresponding to serotypes A, B, C, E and F, and denoted as rBoNT/A(0), rBoNT/B(0), rBoNT/C(0), rBoNT/E(0), and rBoNT/F(0). As a result of being catalytically inactive, these molecules were not able to cleave their respective (SNARE) protein substrates.
A motor neuron-like hybrid cell line (NSC34 cells) (Tebu-Bio, Cedarlane laboratories, France) was cultured on poly-D-lysine coated black multiwells at 5000 cell/well and cultured in DMEM with added 10% FCS and penicillin/streptomycin. After plating, cells were differentiated into motor neurons by exposure to 1 uM retinoic acid and low serum for 4 days, then cells were treated with rBoNT/A(0), rBoNT/B(0), rBoNT/C(0), rBoNT/E(0) and rBoNT/F(0) at 3 different concentrations: 0.1, 1 and 10 nM for 4 days and fixed with paraformaldehyde 4%-sucrose 4%. Brain-derived neurotrophic factor (BDNF) (commercially available from ReproTech EC Ltd, London, UK) 1 ng/mL was used as a positive control of neuronal outgrowth. Cells were fixed with paraformaldehyde 4%-sucrose 4%, then stained with appropriate antibodies. In particular, Anti-8111 Tubulin mAb (Promega G7121) was diluted (1:1000) in 1×PBS+2% BSA+0.3% TritonX-100 and plates were incubated at 37° C. for 3 hours. Alexa Fluor 488 Goat anti-Mouse IgG (H+L) Secondary Antibody (Life Tech cat. A-11001) was then administered (1:2000 in 1×PBS+2% BSA+0.3% TritonX-100) for 1 h at 37° C. Nuclei were stained with DAPI. Image analysis: 6 images per well were taken with ArrayScan XTI HCA Reader (Thermo Fisher Scientific) with a 10× objective. All analysis was performed using Image J software (open source software from NIH, Maryland, USA). Three, independent experiments were carried out. Each independent experiment contained 6 replicates.
Cells were exposed to the different catalytically inactive BoNT serotypes for 4 days (
Thus, these data confirm that the neurotrophic properties of BoNT/A can also be extrapolated to other BoNT serotypes.
Catalytically inactive botulinum toxin rBoNT/A(0) was recombinantly expressed in E. coli. Fragments of BoNT/A were also expressed in E. coli, and are denoted as light chain (L/A), light-chain and translocation domain (LHN/A), and the cell binding domain fragment (HC/A) of the heavy chain. NSC34 cells were exposed to the BoNT/A fragments as well as full-length rBoNT/A(0) as for Example 1.
Similarly to rHC/A, both rL/A and rLHN/A were found to increase neurite length per NSC34 cell at every concentration when compared to an untreated control, similarly to positive control BDNF. It was particularly unexpected that the rL/A and rLHN/A fragments were neurotrophic, since both lack the clostridial toxin receptor binding domain (present in rHC/A).
NSC34 cells were differentiated, then cultured for 4 days under the following experimental conditions: (1) Untreated cells control: cells underwent the same number of manipulations i.e. washes/feeding as compound treated cells however untreated control cells to be exposed to growth medium only, (2) BDNF— positive assay control, 1 ng/ml, (3) BoNT/A(0) at 3 doses (0.1, 1 and 10 nM), (4) Negative assay controls (protein controls): 1. A7030, Sigma, Bovine Serum Albumin (BSA), 2. NBP1-37082, Bio-techne, Recombinant Human Annexin A4 Protein, 3. U-100AT, Bio-techne, Recombinant Plant Ubiquitin Protein, 4. E. coli expression lysate, which does not contain botulinum neurotoxins or fragments thereof. All negative control proteins were tested at 1.5 ug/ml final concentration. This concentration corresponds to 10 nM of BoNT/A(0). Protein solutions were in PBS, except annexin 4-20 mM Tris-HCl buffer (pH8.0) containing 20% glycerol, 0.2M NaCl. All protein solutions were at 1 mg/ml. Cells were stained with Anti-Beta III Tubulin diluter 1:1000 in 1×PBS-4% BSA-0.3% TritonX100 and secondary antibody anti-mouse Alexa Fluor 488; DAPI was used as nuclear stain. All original images of beta 3-tubulin signal were processed using NeurphologyJ (an Image J macro, NIH, Maryland, USA).
Cells were exposed to the different experimental conditions.
A study was designed to investigate the efficacy of catalytically inactive botulinum toxin rBoNT/A(0) in enhancing functional restoration and neuroregeneration using an in vivo mouse dorsal column lesion model. The model is useful for analysing the efficacy of molecules that cause local sprouting and/or long tract axon regeneration. As is well established, crushing injuries are a frequent scenario in spinal cord injury and therefore the model mimics most of the pathological changes that occur in the spinal cord after trauma (see Lagord et al, 2002; Molecular and Cellular Neuroscience 20:69; Esmaelli et al., 2014; Neural Regeneration Research 9:1653; Surey et al., 2014; Neuroscience 275C:62; Almutiri et al., 2018; Scientific Reports 8:10707 for details of the model and the injury responses).
Before surgery, C57/BL mice were injected subcutaneously with Buprenorphine and anaesthetised using 5% of Isoflurane in 1.8 m1/I of 02 with body temperature and heart rate monitored throughout surgery. After partial laminectomy at thoracic level 8 (T8) the ascending sensory, descending motor and segmental proprioceptive axons (SPA) of the spinal dorsal column (SDC) were crushed bilaterally using calibrated watchmakers' forceps 1 mm deep×1 mm wide.
rBoNT/A(0) administration was by way of a single intrathecal 10 μl injection (into the CSF of the spinal canal) of one of 3 doses (100 pg, 100 ng and 50 μg/mouse) at the time of surgery. Treatment groups for each of the 3 doses were as follows:
1. Vehicle (phosphate buffered saline [PBS]), i.e. SDC lesion plus an immediate single 10 μl intrathecal injection of vehicle; n=6 mice.
2. BoNT treated, i.e. SDC lesion plus an immediate single 10 μl intrathecal injection of one of 3 doses of BoNT (100 pg, 100 ng and 50 μg/mouse); 3×n=6/group; 18 mice.
Intrathecal injection of BoNT was carried out as follows. Mice were placed in the prone position and an injection made between L5 and 51 spinal vertebrae. The spinous processes were incised and reflected rostrally to reveal the ligamentum flavum and a blunt 25 G needle was inserted through the ligamentum flavum at an angle of 60° horizontal and access to the intrathecal space was confirmed by reflux of cerebrospinal fluid (CSF) and the presence of a ‘tail flick’. Then 10 μl of injectate was slowly injected over 1 min and CSF expression was facilitated by gentle tail elevation.
1. Locomotor function was measured using the horizontal ladder walking test at baseline (prior to injury) then again at 2 d, 1 w, 2 w, 3 w and 4 w after SDC injury.
2. Qualitative histological assessment at the 4 w time-point of sprouting and regeneration from motor and sensory neurons/axons, i.e. axonal growth over short (<1 mm) and long (˜5 mm) distances. Tissue sections stained for Neurofilament 200 (NF200) detects mature axons. Phosphorylated MAP1b is present in growing axons and growth cones where it maintains a dynamic balance between cytoskeletal components and regulates the stability and interaction of microtubules and actin to promote axonal growth, neural connectivity and regeneration in the central nervous system. MAP1b staining reveals areas of active axonal sprouting.
This tests locomotor function and is performed on a 0.6 metre long horizontal ladder with a width of 8 cm and randomly adjusted rungs with variable gaps of 1-2 cm. Prior to injury, then again at 2 d, 1 w, 2 w, 3 w and 4 w after SDC injury, mice were assessed traversing the ladder and the left and right rear paw slips were recorded along with the total number of steps by an individual unaware of the treatment group. To calculate the mean error rate, the number of slips was divided by the total number of steps.
At 4 w after SDC lesion, mice were intracardially perfused with 4% formaldehyde (Raymond A Lamb, Peterborough, UK) and dissected segments of T8 cord containing the DC injury sites (lesion site+5 mm either side) together with the Tibialis Cranialis muscles were post-fixed for 2 h at RT, cryoprotected in a graded series of sucrose, blocked up in optimal cutting temperature medium (OCT; Raymond A Lamb) and sectioned at 15 μm thick using a Bright cryostat.
Sections were thawed at room temperature for 30 min before washing twice in 0.1M phosphate buffered saline, pH7.4 (PBS; Raymond A Lamb). Sections were then permeablised in 0.1% Triton X-100 in PBS (Sigma) for 10 min and blocked in PBS containing 0.5% bovine serum albumin (BSA) and 0.1% Triton-X100 (all from Sigma) for 30 min at room temperature. Sections were then incubated with the appropriate primary antibody diluted with antibody diluting buffer (ADB; PBS containing 0.5% BSA and 0.05% Tween-20 (all from Sigma)) and incubated overnight at 4° C. in a humidified chamber. Sections were then washed in PBS and incubated with appropriate fluorescently-labelled secondary antibody diluted in ADB. Sections were then washed in PBS and coverslips mounted using Vectashield containing DAPI (Vector Laboratories, Peterborough, UK). Negative controls were included in each run that included omission of primary antibody and these were used to set the background threshold levels for image capture. Sections were viewed and images captured using an Axioplan 2 epifluorescent microscope equipped with an Axiocam HRc running Axiovision software.
Primary antibodies used were as follows:
Secondary antibodies used were as follows:
Statistical analyses on the functional data were performed using SPSS 20 (IBM, USA). Normal distribution tests were carried out to determine the most appropriate statistical analysis to compare treatments. Statistical significance was determined at p<0.05.
The immunohistochemical assessment employed the use of antibodies to Neurofilament 200 (NF200) and MAP1b. Neurofilament 200 (NF200) is expressed in mature axons and the pMAP1b antibody reveals neurofilaments in the terminals of actively sprouting axons, illustrating axons that are still actively sprouting around and within the lesion site.
The rapidity of the onset of improvement in performance in the functional test shows that rBoNT/A(0) caused axonal sprouting with the establishment of useful functional synapses below the lesion. Qualitative immunohistochemistry provided evidence of BoNT-induced florid axonal sprouting locally through the SDC lesion site.
These in vivo data are clear evidence validating a role for rBoNT/A(0) in the treatment of neurological disorders.
A number of full-length catalytically-inactive recombinant BoNT serotypes, as well as BoNT fragments, and variants were tested for their modulatory action on neurite outgrowth in vitro.
Cells exposed to the polypeptides were compared to those exposed to a positive control (1 ng/ml BDNF). Mouse Motor Neuron-Like Hybrid (NSC34) cells were differentiated and exposed during 4 days in vitro (DIV) to different polypeptides at 3 different doses (0.1 nM, 1 nM, and 10 nM).
NSC34 cells were produced by fusion of motor neuron enriched, embryonic mouse spinal cord cells and mouse neuroblastoma (Cashman et al. Dev Dyn. 1992 July; 194(3):209-21, which is incorporated herein by reference). Said cells mimic many properties of motor neurons, including choline acetyltransferase, acetylcholine synthesis, storage and release and neurofilament triplet proteins. Moreover, NSC34 spinal cord motor neurons express glutamate receptor proteins and generate action potentials. NSC34 neurons have been widely used to study mechanisms of neuron signalling and neuron degeneration.
The following experimental scheme was adopted: Screening on Neuronal cell line (NSC34):
NSC34 cells were cultivated on poly-D-lysine-coated glass coverslips in DMEM plus 10% FCS.
After plating, cells were differentiated into motor neurons by exposure to retinoic acid and low serum levels for 4 days. Cells were cultured either in the presence/absence of the polypeptides at a specific timepoint. (i.e. 4 DIV). Test data was compared with effects seen on positive (BDNF) and also negative (BSA) control data.
After 4 days in vitro (DIV), cells were fixed in 4% paraformaldehyde, stained with specific neuronal markers (beta tubulin) and quantitatively assayed for neurite outgrowths (neurite extension, axonal elongation, arborization). Image acquisition was carried out using Operetta CLS HCS microscope (PerkinElmer) by means of a 20× objective. Per each well, six (6) fields-of-view were acquired. The neurite outgrowth analysis was performed and the mean neurites per cell assessed.
For BoNT/A, the LHN/A fragment (light-chain plus translocation domain) had improved activity compared to the cell binding domain (HC domain) fragment (see
For both BoNT/FA and BoNT/F, the LHN and LC (light-chain only) fragments showed improved activity compared to the HC domain fragments (see
Finally, the variant HC domain fragments were all shown to be highly efficacious (
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1914034.2 | Sep 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2020/052363 | 9/30/2020 | WO |
