Clostridial neurotoxins containing activated endosomal protease cleavage sites.

Abstract

The present invention relates to clostridial neurotoxins modified to contain an endosomal protease cleavage site within the activation loop, where cleavage at the site produces an active di-chain clostridial neurotoxin. The invention also relates to methods for producing the clostridial neurotoxins, as well as related pharmaceutical compositions, nucleotide sequences, and therapeutic and cosmetic uses. The invention further relates to methods for proteolytically cleaving the single-chain clostridial neurotoxins to the corresponding di-chain clostridial neurotoxins.

Description

[Technical Field]

[0001] The present invention relates to clostridial neurotoxins modified to contain an endosomal protease cleavage site within the activation loop, where cleavage at the site produces an active two-chain clostridial neurotoxin. The invention also relates to methods for producing the clostridial neurotoxins, as well as related pharmaceutical compositions, nucleotide sequences, and therapeutic and cosmetic uses. The invention further relates to methods for proteolytically cleaving the single-chain clostridial neurotoxins to the corresponding two-chain clostridial neurotoxins. [Background technology]

[0002] Bacteria of the genus Clostridium produce highly potent and specific protein toxins that can harm neurons and other cells to which they are delivered. Examples of such clostridial neurotoxins include those produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes A-G and X (see WO2018 / 009903A2), as well as those produced by C. baratii and C. butyricum.

[0003] Clostridial neurotoxins are some of the most potent toxins known. For example, the median lethal dose (LD) of botulinum neurotoxin in mice is 50 ) ranges 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. Botulinum toxin acts at the neuromuscular junction to inhibit cholinergic transmission in the peripheral nervous system, while tetanus toxin acts in the central nervous system.

[0004] Clostridial neurotoxins are expressed in Clostridium as single-chain polypeptides. Each clostridial neurotoxin has a catalytic light chain, which is separated from the heavy chain (which includes an N-terminal translocation domain and a C-terminal receptor-binding domain) by an exposed region called the activation loop. During protein maturation, proteolytic cleavage of the activation loop separates the light and heavy chains of the clostridial neurotoxin, which are held together by disulfide bridges to generate the fully active dichain toxin.

[0005] This activation process must be replicated during the production of standard recombinant toxins. Exogenous proteases with well-defined cleavage motifs, such as trypsin or Lys-C, are used in conventional production methods to proteolytically activate single-chain clostridial neurotoxins. However, for some clostridial neurotoxins, incubation with Lys-C or trypsin results in partial or inappropriate cleavage of the single-chain polypeptide, resulting in the production of contaminating single-chain and / or inactive cleavage / degradation products (e.g., in the case of BoNT / E). For example, activation is problematic for botulinum neurotoxin serotype X (BoNT / X, see WO 2018 / 009903 A2), in which cleavage with trypsin or Lys-C completely degrades the polypeptide. Therefore, at present, no universal exogenous protease exists for activating clostridial neurotoxins. This is particularly problematic when identifying new clostridial neurotoxins or producing modified (e.g., chimeric or hybrid) neurotoxins, which require screening multiple proteases to determine proper activation. For retargeted clostridial neurotoxins, some standard proteases used for activation may also cleave within the exogenous targeting site, resulting in misprocessed protein with reduced targeting to the desired cell type. To avoid such off-target cleavage, alternative targeting sites must be identified (which may not always be possible) or the targeting site must be designed to eliminate the cleavage site in the standard protease, which may negatively affect the structure of the targeting site and / or increase the cost of design and production.

[0006] Furthermore, in vitro activation of clostridial neurotoxins has many drawbacks. The use of exogenous proteases (especially GMP-grade proteases) and their removal after activating the clostridial neurotoxin are costly. Reliance on one or a limited number of suppliers of GMP-grade proteases may create weaknesses in the supply / production chain. Purification of activated clostridial neurotoxins from activated exogenous proteases may also affect production efficiency and yield. In addition, producing active dichain clostridial neurotoxins according to conventional production methods requires strict safety and control procedures, which also increase production costs and time. Strict safety precautions are also required for physicians using active dichain clostridial neurotoxins.

[0007] The present invention overcomes one or more of the problems set forth above. Summary of the Invention

[0008] Endosomes are small membrane-bound vesicles within eukaryotic cells. They are responsible for transporting internalized materials from outside the cell. Some endosomes maintain an acidic pH, which allows them to dissociate proteins from receptors. Once separated, they can transport these different components. Some molecules are targeted for recycling back to the cell surface, while others fuse with lysosomes, where their contents are degraded by hydrolase enzymes. Endosomes also transport materials to and from the Golgi and between the apical and basal polarized compartments within the cell. Endosomes contain numerous proteases that degrade internalized proteins.

[0009] The present inventors have previously demonstrated that inserting a furin cleavage site into the activation loop of a clostridial neurotoxin allows the clostridial neurotoxin to be activated in vivo (see PCT Application No. PCT / GB2022 / 050756, incorporated herein by reference in its entirety). This represents a paradigm shift in terms of the production, processing, activation, and actual therapeutic use of clostridial neurotoxins. Specifically, to the extent that previous attempts in the art have been made to introduce exogenous cleavage sites into clostridial neurotoxins, the goal has always been to facilitate the in vitro production and processing of clostridial neurotoxins, with the resulting neurotoxins being administered in a di-chain form. Here, we have demonstrated that the potential for in vivo activation of this clostridial neurotoxin is not limited to the use of furin, but that other endogenous proteases, particularly endosomal proteases (e.g., cathepsin L and asparaginyl endopeptidase (AEP)), can also cleave appropriate cleavage sites exogenously introduced into the clostridial neurotoxin.

[0010] Additionally, the endosomal protease-activated modified clostridial neurotoxins of the present invention offer several potential advantages over conventionally activated clostridial neurotoxins, such as improved safety for operators (e.g., clinicians handling the endosomal protease-activated modified neurotoxins of the present invention for administration to patients and workers involved in the production of the endosomal protease-activated modified neurotoxins) and / or reduced manufacturing burden / cost. Also, the endosomal protease-activated modified neurotoxins of the present invention may have an enhanced safety profile for patients.

[0011] Thus, for the first time, the inventors provide a single-chain clostridial neurotoxin (e.g., a modified BoNT / A1 with an endosomal protease cleavage site with therapeutic potential) that does not require activation to a two-chain form prior to administration.

[0012] Thus, the present invention provides a modified clostridial neurotoxin that includes an endosomal protease cleavage site, wherein cleavage at the cleavage site results in production of a di-chain form of the modified clostridial neurotoxin. The endosomal protease cleavage site can be a cleavage site specific for (a) aspartic endopeptidase (AEP), or (b) a cathepsin, optionally cathepsin L1, B, D, K, or S. The endosomal protease cleavage site can comprise or consist of (a) an APE core motif selected from SEQ ID NOs: 208-214, (b) a cathepsin L core motif selected from SEQ ID NOs: 138 and / or 188-197, (c) a cathepsin B core motif selected from SEQ ID NOs: 20, 181, 198, and / or 199, and / or (d) a cathepsin D core motif selected from SEQ ID NOs: 200-207. Endosomal protease cleavage sites are shown in SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 20 The modified clostridial neurotoxin may comprise or consist of one or more of SEQ ID NOS: 42, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, and / or 187. The modified clostridial neurotoxin may comprise an exogenous activation loop comprising or consisting of any one of SEQ ID NOS: 35, 36, 37, 38, 127, 128, and / or 129. The endogenous activation loop of a clostridial neurotoxin, or a portion thereof, may be replaced with one or more endosomal protease cleavage sites in accordance with the present invention. The endogenous neurotoxin activation loop may be one or more selected from SEQ ID NOS: 89-112.

[0013] The clostridial neurotoxin can be (a) botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F, serotype G, or serotype X, or tetanus neurotoxin (TeNT), or (b) a chimeric or hybrid BoNT. Preferably, the clostridial neurotoxin is BoNT / X, BoNT / A (e.g., BoNT / A1), or BoNT / B. In a particularly preferred embodiment, the clostridial neurotoxin is BoNT / X.

[0014] The modified clostridial neurotoxin may be a single-chain clostridial neurotoxin (a) encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site optionally selected from SEQ ID NOs: 165, 166, and 167, and / or (b) comprising a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is replaced by at least one endosomal protease cleavage site or an extrinsic activation loop (optionally selected from SEQ ID NOs: 127-129).

[0015] The modified clostridial neurotoxin is a clostridial neurotoxin that inhibits the endogenous H C or H CC may be a retargeted Clostridial neurotoxin in which the targeting moiety (TM) is replaced by an exogenous targeting moiety (TM). Preferably, the modified Clostridial neurotoxin may be a retargeted BoNT / X or BoNT / A.

[0016] The present invention also provides a modified retargeted BoNT / X containing an endosomal protease cleavage site, comprising a polypeptide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity to SEQ ID NOs: 160-162.

[0017] The present invention further provides a method for proteolytically cleaving a modified clostridial neurotoxin according to the present invention into the corresponding di-chain clostridial neurotoxin, which method comprises contacting the modified clostridial neurotoxin with an endosomal protease specific for an endosomal protease cleavage site to produce the di-chain clostridial neurotoxin.

[0018] The present invention also provides a di-chain Clostridial neurotoxin obtainable by the method.

[0019] The present invention further provides polynucleotides encoding modified Clostridial neurotoxins according to the invention.

[0020] The present invention also provides an expression vector comprising a polynucleotide as defined in claim 15 operably linked to a promoter.

[0021] The polynucleotide or expression vector may comprise or consist of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site optionally selected from SEQ ID NOs: 165, 166, and 167, and / or (b) encodes a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is replaced by at least one endosomal protease cleavage site or extrinsic activation loop (optionally selected from SEQ ID NOs: 127-129).

[0022] The present invention also provides a method for producing a modified Clostridial neurotoxin of the present invention, the method comprising the steps of expressing a polynucleotide or expression vector of the present invention in a cell and recovering the expressed modified Clostridial neurotoxin, and the method may further comprise the step of introducing the polynucleotide or expression vector of the present invention into a cell.

[0023] The present invention further provides a cell expressing a modified Clostridial neurotoxin of the present invention, which cell may comprise a polynucleotide or expression vector of the present invention.

[0024] The present invention further provides pharmaceutical compositions comprising a modified Clostridial neurotoxin of the present invention or a two-chain Clostridial neurotoxin of the present invention and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, propellant, and / or salt.

[0025] The present invention also provides a modified Clostridial neurotoxin, a double-chain Clostridial neurotoxin, or a pharmaceutical composition of the present invention for use in a method for preventing or treating a disease or disorder indicating a need for treatment with a botulinum neurotoxin, and optionally the disease or disorder is a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, or the like). stonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapeutic (cosmetic) applications that benefit from cell / muscle inactivation (via SNARE downregulation or inactivation), neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, vertical strabismus, lateral rectus palsy, nystagmus, myopathy due to thyroid abnormalities), writer's cramp, teeth grinding, Wilson's disease, tremors, tics, segmental myoclonus, spasticity, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder abnormalities, animus, back spasms, muscle spasms or rigidity (charley horse), levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremor, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), forehead grooves, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary neuropathy, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders. Preferably, the composition of the present invention may be used for the prevention or treatment of a disease or condition selected from limb spasticity (upper or lower limb), cervical dystonia, headache disorders (preferably migraine), blepharospasm, hemifacial spasm, and lower urinary tract disorders (e.g., bladder pain syndrome (preferably interstitial cystitis)), overactive bladder, and detrusor overactivity (e.g., neurogenic detrusor overactivity).

[0026] The present invention also provides for the use of a modified Clostridial neurotoxin, a double-chain Clostridial neurotoxin, or a pharmaceutical composition of the present invention in the manufacture of a medicament for preventing or treating a disease or disorder indicating a need for treatment with a botulinum neurotoxin, optionally wherein the disease or disorder is a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapeutic (cosmetic) applications that benefit from cell / muscle inactivation (via SNARE downregulation or inactivation), neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, vertical strabismus, lateral rectus palsy, nystagmus, myopathy due to thyroid abnormalities), writer's cramp, bruxism, Wilson's disease, tremors, tics, segmental myoclonus, spasticity, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder control disorders, animus, back spasms, muscle spasms or rigidity (charley horse), levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremor, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), forehead grooves, skin wrinkles, cancer, uterine disorders, genitourinary disorders, genitourinary neuropathy, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders. Preferably, the composition of the present invention may be used for the prevention or treatment of a disease or condition selected from limb spasticity (upper or lower limb), cervical dystonia, headache disorders (preferably migraine), blepharospasm, hemifacial spasm, and lower urinary tract disorders (e.g., bladder pain syndrome (preferably interstitial cystitis)), overactive bladder, and detrusor overactivity (e.g., neurogenic detrusor overactivity).

[0027] The modified Clostridial neurotoxin (e.g., within a pharmaceutical composition) can be administered to a subject in a single-chain form. The Clostridial neurotoxin or pharmaceutical composition can be substantially free of the double-chain form of the Clostridial neurotoxin. The Clostridial neurotoxin or pharmaceutical composition can contain less than 400 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 300 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 200 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 100 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 50 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin.

[0028] The present invention also provides a cosmetic composition comprising a modified clostridial neurotoxin or a di-chain clostridial neurotoxin of the present invention and a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant, and / or salt.

[0029] The present invention further provides use of the cosmetic composition of the present invention to prevent or alleviate cosmetic indications requiring the application of a botulinum neurotoxin. The clostridial neurotoxin can be administered to a subject in a single-chain form. The clostridial neurotoxin or cosmetic composition can be substantially free of a double-chain form of the clostridial neurotoxin. The clostridial neurotoxin or cosmetic composition can contain less than 400 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 300 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 200 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 100 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 50 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin.

[0030] The present invention also provides a method for proteolytically cleaving a single-chain Clostridial neurotoxin into a corresponding di-chain Clostridial neurotoxin, the method comprising: (a) providing a single-chain Clostridial neurotoxin; and (b) contacting the single-chain Clostridial neurotoxin with an endosomal protease, wherein the single-chain Clostridial neurotoxin has an activation loop that comprises or consists of a polypeptide sequence defined herein, and the endosomal protease hydrolyzes the peptide bond of the activation loop to produce the di-chain Clostridial neurotoxin. The single-chain clostridial neurotoxin may (a) be a modified clostridial neurotoxin of the present invention; (b) be encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site optionally selected from SEQ ID NOs: 165, 166, and 167; and / or (c) comprise a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is replaced by at least one endosomal protease cleavage site or an extrinsic activation loop (optionally selected from SEQ ID NOs: 127-129). [Brief explanation of the drawings]

[0031] [Figure 1] (A) Coomassie staining of purified untreated CatDReo (BIO4934), Ebo (BIO4935), CatBL (BIO4945), and AEP (BIO4938) at pH 5 and 7.2. (B) Western blot analysis of purified untreated CatDReo (BIO4934), Ebo (BIO4935), CatBL (BIO4945), and AEP (BIO4938) at pH 5 and 7.2 using αLC / A antibody. [Figure 2A]Coomassie staining (left panel), Western blot analysis using αLC / A antibody (center panel), and Western blot analysis using αHis antibody (right panel) of CatDReo (BIO4934) incubated with serial dilutions of DTT-reduced cathepsin L1 and resolved by SDS-PAGE. [Figure 2B] Coomassie staining (left panel), Western blot analysis using αLC / A antibody (center panel), and Western blot analysis using αHis antibody (right panel) of Ebo (BIO4935) incubated with serial dilutions of DTT-reduced cathepsin L1 and resolved by SDS-PAGE. [Figure 3] (A) Coomassie staining, (B) Western blot analysis using αLC / A antibody, and (C) Western blot analysis using αHis antibody of CatBL (BIO4945) incubated with serial dilutions of DTT-reduced cathepsin B and resolved by SDS-PAGE. [Figure 4] (A) Coomassie staining, (B) Western blot analysis using αLC / A antibody, and (C) Western blot analysis using αHis antibody of AEP (BIO4938), incubated with serial dilutions of DTT-reduced AEP and resolved by SDS-PAGE. DETAILED DESCRIPTION OF THE INVENTION

[0032] definition Unless otherwise defined, 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 a dictionary of many of the terms used in this disclosure for those of ordinary skill in the art. The meaning and scope of the terms should be clear, but in the event of any potential ambiguity, the definitions set forth herein take precedence over any dictionary or external definitions.

[0033] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. Specifically, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure.

[0034] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments of and examples of the present disclosure have been described herein for illustrative purposes; however, as those skilled in the relevant art will recognize, various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions may be presented in a given order, alternative embodiments may perform the functions in a different order or perform the functions substantially simultaneously. The teachings of the present disclosure presented herein may be applied to other procedures or methods, as appropriate. The various embodiments described herein may be combined to yield further embodiments. Aspects of the present disclosure may be modified, as appropriate, to employ the structure, function, and concepts of the above-mentioned references and applications to provide further embodiments of the present disclosure. Furthermore, in light of biological functional equivalents, protein structures may be modified somewhat without affecting biological or chemical activity in kind or amount. These and other modifications may be made to the present disclosure in light of the above detailed description. All such modifications are intended to be within the scope of the appended claims.

[0035] Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation; any amino acid sequence is written left to right in amino to carboxy orientation, respectively.

[0036] The headings provided herein do not limit the various aspects or embodiments of the disclosure.

[0037] As used herein, the term "capable of" when used with a verb encompasses or implies the action of the corresponding verb. For example, "capable of interacting" also means "interacting," "cleavable" also means "cleaving," "capable of binding" also means "binding," and "specifically targetable" also means "specifically target."

[0038] Numerical ranges are inclusive of the numerical endpoints defining the range. When a range of values ​​is disclosed, it is understood that each intervening value (to the nearest tenth of the lower limit between the upper and lower limits of that range, unless the context clearly dictates otherwise) is also expressly disclosed. Each subrange between any stated or intervening value within a stated range and any other stated or intervening value is encompassed within the disclosure. The upper and lower limits of these subranges may independently be included or excluded within the subrange, and each range in which one, neither, or both of the upper and lower limits are included within the disclosure, subject to any specifically excluded limits within the stated range. When a stated range includes one or both of the upper and lower limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0039] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-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 cases, the term "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. In this disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be encoded by multiple nucleotide sequences due to the degeneracy of the genetic code.

[0040] A "fragment" of a polypeptide typically comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or more of the original polypeptide.

[0041] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA, or alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one embodiment, the nucleic acid can be DNA. In another embodiment, the nucleic acid can be RNA. A preferred nucleic acid molecule is DNA, including genomic DNA or cDNA. Another preferred nucleic acid molecule is RNA (including siRNA, shRNA, and antisense oligonucleotides).

[0042] As defined herein, the term "endosome" refers to a membrane-bound intracellular organelle. Endosomes are typically part of the endocytic membrane trafficking pathway that originates from the trans-Golgi network. The term "endosome" encompasses early endosomes, late endosomes, and recycling endosomes, unless expressly stated to the contrary. The term "endosome" may also encompass lysosomes and / or other intracellular vesicles. Each of these "early," "late," "recycling," and "lysosomal" compartments is characterized by distinct protein-lipid composition, morphology, and intraluminal pH.

[0043] Early endosomes typically have a diameter of up to 1 μm (e.g., 100–500 nm) and may be connected by tubules of approximately 50 nm in diameter. Markers of early endosomes include RAB5A and RAB4, RAB11, transferrin, and early endosomal antigen 1 (EEA1).

[0044] Late endosomes are typically spherical and do not have connecting tubules. Late endosomes may contain multiple dense intraluminal vesicles. Markers for late endosomes include RAB7, RAB9, and the mannose 6-phosphate receptor. Late endosomal membranes (and lysosomes) may contain a substance called lysobisphosphatidic acid (LBPA). As endosomes mature, they become more acidic; typically, late endosomes have a lower pH (approximately 5.0) than early endosomes (approximately 6.5).

[0045] Recycling endosomes are concentrated at microtubule-organizing centers and consist primarily of tubular networks. Markers for recycling endosomes include RAB11 and RAB4.

[0046] Lysosomes are vesicles derived from the Golgi apparatus that contain up to 50 different degradative enzymes. Lysosomes typically have the lowest pH of any intracellular vesicular compartment (approximately pH 4.5-5.0), and this acidic pH is necessary for the enzymes within them to function. Lysosomal markers include highly glycosylated lysosome-associated membrane proteins (LAMPs), such as LAMP-1 and LAMP-2, and RAB9.

[0047] As used herein, the term "spacer" refers to a flexible peptide used in an exogenous activation loop or modified BoNT / C activation loop, or used with an exogenous protease cleavage site, and is typically included to maintain the secondary structure of the exogenous activation loop in the modified clostridial neurotoxins of the present invention. Spacers used in the modified clostridial neurotoxins of the present invention can comprise an amino acid sequence of 1 to 30 amino acid residues (e.g., 5 to 30 amino acid residues, 10 to 25 amino acid residues, or about 5 to about 20 amino acid residues). Spacers can comprise or consist of small amino acid residues, such as glycine, threonine, arginine, serine, asparagine, glutamine, alanine, aspartic acid, proline, glutamic acid, lysine, leucine, and / or valine, particularly glycine, serine, alanine, leucine, and / or valine. Spacers comprising or consisting of glycine, serine, and / or alanine are preferred, with glycine and serine being particularly preferred. Thus, the most commonly used spacer has a sequence consisting primarily of a string of Gly and Ser residues (the "GS" linker), which is (Gly-Gly-Gly-Gly-Ser). n (SEQ ID NO: 153). Non-limiting examples of GS linkers include GS5 or (GGGGS)1 (SEQ ID NO: 154), GS10 or (GGGGS)2 (SEQ ID NO: 155), GS15 or (GGGGS)3 (SEQ ID NO: 156), GS20 or (GGGGS)4 (SEQ ID NO: 157), and GS25 or (GGGGS)5 (SEQ ID NO: 158).

[0048] As used herein, the term "core motif" refers to the minimal amino acid sequence that can be cleaved by a given endosomal protease. As a non-limiting example, the core motif in cathepsin L defines the minimal amino acid sequence that can be cleaved by cathepsin L. For any given endosomal protease, there may be multiple core motifs.

[0049] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. The terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100%, or any increase between 10 and 100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0050] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant decrease. The terms "reduce," "reduction," "reduce," or "inhibit" typically mean a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" encompasses complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition (i.e., abolition) compared to a reference level.

[0051] Other term definitions may appear throughout the specification. Before describing exemplary embodiments in more detail, it is to be understood that the present disclosure is not limited to particular embodiments described, 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, as the scope of the present disclosure will be defined only by the appended claims.

[0052] It should 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, a reference to "a clostridial neurotoxin" includes a plurality of such candidate agents, a reference to "the clostridial neurotoxin" includes a reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so on. Furthermore, the use of "including" as well as other forms (e.g., "includes" and "included") is not limiting.

[0053] "About" can generally refer to the degree of error allowed for in the measured quantity, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. Preferably, the term "about" is understood herein as plus or minus (±) 5% of the numerical value with which it is used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0054] The term "consisting of" refers to the compositions, methods, and their respective components described herein, and excludes any elements not recited in the description of the invention.

[0055] As used herein, the term "consisting essentially of" refers to elements necessary for a given invention. The term permits the presence of elements (i.e., inert or non-immunogenic components) that do not materially affect the basic and novel or functional characteristic(s) of the invention.

[0056] An embodiment described as "comprising" one or more features may also be considered as disclosing corresponding embodiments that "consist" of and / or "consist essentially of" such features.

[0057] The term "deletion," as used herein, refers to the removal of one or more amino acid residues of a polypeptide without the replacement of one or more amino acid residues at the site of the deletion. Thus, for example, if one amino acid residue is deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide will have x-1 amino acid residues.

[0058] The term "indel," as used herein, refers to the deletion of one or more amino acid residues in a polypeptide, with a different number of amino acid residues (more or fewer) inserted at the site of the deletion compared to the number of deleted amino acid residues. Thus, for example, in the case of an indel in which two amino acid residues are deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide will have x-1 amino acid residues, or x+1 or more amino acid residues. Insertions and deletions can be made in any order, either sequentially or simultaneously.

[0059] The term "substitution," as used herein, refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same positions. Thus, in the case of a substitution in a polypeptide sequence having (for example) x number of amino acid residues, the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

[0060] The term "insertion," as used herein, refers to the addition of one or more amino acid residues to a polypeptide without deletion of one or more amino acid residues of the polypeptide at the insertion site. Thus, (for example) if one amino acid residue is inserted into a polypeptide sequence having x number of amino acid residues, the resulting polypeptide will have x+1 amino acid residues.

[0061] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format, with the understanding that this range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​expressly recited as the limits of the range, but also all individual numerical values ​​or subranges encompassed within that range, as if each numerical value or subrange were expressly recited.

[0062] The individual may be an individual who has previously been diagnosed or identified as suffering from or having a condition requiring treatment, or one or more complications associated with such a condition, and optionally has already received treatment for the condition defined herein or one or more complications associated with the condition. Alternatively, the individual may be an individual who has not previously been diagnosed with a condition defined herein or one or more complications associated with the condition. For example, the individual may be an individual who exhibits one or more risk factors for a condition, or one or more complications associated with the condition, or may be a subject who does not exhibit risk factors.

[0063] An "individual in need" of treatment for a particular condition can be an individual who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0064] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammalian individual. An "individual" can be any mammal. Generally, an individual can be a human; in other words, in one embodiment, an "individual" is a human. An "individual" can be an adult, a minor, or an infant. An "individual" can be male or female.

[0065] The term "pharmaceutically acceptable," as used herein, means approved by a regulatory agency of a federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0066] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications are prior art to the claims appended hereto.

[0067] Modified Clostridial Neurotoxins The present invention provides modified clostridial neurotoxins that contain an endosomal protease cleavage site. Typically, cleavage at the endosomal protease cleavage site results in the production of a di-chain form of the modified clostridial neurotoxin. In other words, cleavage at the endosomal protease cleavage site results in the activation of the modified clostridial neurotoxin. The endogenous (native) activation loop of a clostridial neurotoxin can be replaced (or partially replaced) with an endosomal protease cleavage site. Thus, the term "endosomal protease cleavage site" can be used interchangeably with the term "endosomal protease activation site," and an extrinsic activation loop, as defined herein, typically comprises or consists of one or more endosomal protease cleavage sites as described herein. The modified clostridial neurotoxins of the present invention can be activated in vivo. Thus, modified clostridial neurotoxins open up new areas of processing and therapeutic use for clostridial neurotoxins, allowing the toxins to be produced and administered as single-chain clostridial neurotoxins, which are then cleaved to yield the active two-chain form in vivo.

[0068] Clostridial neurotoxins (before modification) are typically characterized in that their intrinsic activation loops are inefficiently proteolytically processed by one or more endosomal proteases. In contrast to clostridial neurotoxins (before modification), the modified clostridial neurotoxins of the present invention are not inefficiently proteolytically processed by one or more endosomal proteases into which a cleavage site has been introduced, and / or the peptide bonds outside the extrinsic activation loop of the modified clostridial neurotoxin are not hydrolyzed by the one or more endosomal proteases. Thus, clostridial neurotoxins (before modification) are typically resistant to proteolytic processing by one or more endosomal proteases. The terms "inefficiently proteolytically processed by one or more endosomal proteases," "resistant to proteolytic processing by one or more endosomal proteases," "not substantially hydrolyzed by one or more endosomal proteases," "inefficiently activated by one or more endosomal proteases," "resistant to activation by one or more endosomal proteases," and "not substantially activated by one or more endosomal proteases" are used interchangeably herein. Typically, a clostridial neurotoxin (before modification) is resistant to proteolytic processing by one or more endosomal proteases into which a cleavage site(s) has been introduced according to the present invention. A clostridial neurotoxin (before modification) may also be resistant to proteolytic processing by one or more endosomal proteases into which a cleavage site(s) has not been introduced according to the present invention.

[0069] A clostridial neurotoxin (before modification) is typically a neurotoxin whose peptide bonds (either inside or outside the activation loop) are not hydrolyzed or substantially not hydrolyzed by one or more endosomal proteases. The term "substantially not hydrolyzed" means that less than 10%, 5%, 4%, 3%, 2%, or 1% of the clostridial neurotoxin present in the reaction contains peptide bonds that are hydrolyzed by one or more endosomal proteases in the methods of the present invention.

[0070] Thus, a clostridial neurotoxin (before modification) may not contain one or more endosomal protease cleavage sites (e.g., as defined herein, e.g., any one of SEQ ID NOS: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or 12-34, 130-152, or 171-187) within its endogenous activation loop. A clostridial neurotoxin (before modification) may not contain one or more endosomal protease core motifs (e.g., as defined herein, e.g., any one of SEQ ID NOS: 20, 138, 181, and / or 188-214).

[0071] The clostridial neurotoxin (before modification) may not contain one or more endosomal protease cleavage sites (e.g., as defined herein, e.g., any one of SEQ ID NOS: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or 12-34, 130-152, or 171-187) and / or one or more endosomal protease core motifs (e.g., as defined herein, e.g., any one of SEQ ID NOS: 20, 138, 181, and / or 188-214) for an endosomal protease intended to be used to activate the modified clostridial neurotoxin. As a non-limiting example, if a modified clostridial neurotoxin contains a cathepsin L cleavage site, the corresponding pre-modified clostridial neurotoxin may not contain a cathepsin L cleavage site and / or a core motif within its endogenous activation loop. In some embodiments in which one or more endosomal protease cleavage sites include an AEP cleavage site, the (pre-modified) clostridial neurotoxin may be BoNT / B, BoNT / C, or BoNT / F, particularly BoNT / B or BoNT / C. In particularly preferred embodiments, the clostridial neurotoxin is BoNT / X, or a chimera or hybrid of BoNT / X and another clostridial neurotoxin.

[0072] The present invention can involve replacing the endogenous activation loop (or a portion thereof) of any clostridial neurotoxin with one or more endosomal protease (exogenous) cleavage sites described herein, or an exogenous activation loop containing one or more endosomal protease cleavage sites. The clostridial neurotoxin can be a botulinum neurotoxin (BoNT) or a tetanus neurotoxin (TeNT). Preferably, the clostridial neurotoxin is a botulinum neurotoxin, such as BoNT / A, BoNT / B, BoNT / C1, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X, or a chimera or hybrid thereof. In a particularly preferred embodiment, the clostridial neurotoxin is BoNT / X, or a chimera or hybrid thereof.

[0073] The term "intrinsic activation loop," as used herein, refers to an activation loop present in a subject clostridial neurotoxin (e.g., a subject clostridial neurotoxin of the indicated serotype). For example, BoNT / A1 comprises a BoNT / A1 heavy chain and a light chain, and therefore the intrinsic activation loop of BoNT / A1 is the A1 activation loop. For clostridial neurotoxin chimeras or hybrids, one skilled in the art would recognize, for example, the L chain and the H chain. NThe "intrinsic activation loop" can be identified by determining the serotype(s) from which the domain is derived. In some embodiments, a chimeric or hybrid clostridial neurotoxin can have an intrinsic activation loop (a fusion of activation loops from two different serotypes). As an example, a chimeric clostridial neurotoxin (e.g., BoNT / A1C1) has a BoNT / A1 light chain and a translocation domain, and therefore the intrinsic BoNT / A1C1 activation loop is the A1 activation loop. Intrinsic activation loops are typically linked by cysteine ​​residues, which form disulfide bonds covalently linking the light and heavy chains of the (prior to modification) clostridial neurotoxin. Thus, an intrinsic activation loop sequence may be described with or without a bound cysteine ​​residue (as described herein). Those skilled in the art will understand that these definitions can be used interchangeably and will be able to readily identify an intrinsic activation loop with or without a bound cysteine ​​residue.

[0074] Typically, an "intrinsic activation loop" is an activation loop that does not contain or consist of one or more endosomal protease cleavage sites described herein (e.g., SEQ ID NOs: 1-38, 12-38, 1-34, or 12-34). An "intrinsic activation loop" is any activation loop that does not contain or consist of one or more endosomal protease cleavage sites selected from (i) SEQ ID NOs: 1-10, 12-21, or 27-38, (ii) SEQ ID NOs: 12-21 or 27-38, (iii) SEQ ID NOs: 1-10, 12-21, or 17-34, or (iv) SEQ ID NOs: 12-21 or 27-34.

[0075] In contrast, "exogenous activation loop," as used herein, refers to an activation loop that is different from the endogenous activation loop present in a subject clostridial neurotoxin (e.g., a subject clostridial neurotoxin of an indicated serotype), where the exogenous activation loop comprises one or more endosomal protease cleavage sites. For example, the BoNT / C1 activation loop has a different polypeptide sequence from the wild-type BoNT / A1 activation loop, and therefore the BoNT / C1 activation loop is exogenous to BoNT / A1. For clostridial neurotoxin chimeras or hybrids, one skilled in the art can easily distinguish between, for example, the L chain and the H chain. N By determining the serotype(s) from which the domain is derived, it can be determined whether the activation loop is an "exogenous activation loop." For example, if the L chain is a BoNT / BL chain and the H N If the domain is derived from BoNT / D, the intrinsic activation loop can have a portion of the BoNT / B sequence and a portion of the BoNT / D sequence, and if the activation loop (e.g., the C1 activation loop) is different and contains one or more endosomal protease cleavage sites, it is considered an "exogenous activation loop."

[0076] Determining whether an activation loop is an "intrinsic activation loop" can be done by aligning the sequence of a subject clostridial neurotoxin with the activation loop and determining whether an activation loop is present within the sequence of the subject clostridial neurotoxin. If present, the activation loop can be identified as an intrinsic activation loop. As described herein, the intrinsic activation loop of a clostridial neurotoxin has been replaced with an extrinsic cleavage site that is one or more endosomal protease cleavage sites, or an extrinsic activation loop that includes one or more endosomal protease cleavage sites.

[0077] Typically, according to the present invention, one or more endosomal protease cleavage sites are inserted between the two cysteine ​​residues bound to the endogenous activation loop of the unmodified Clostridial neurotoxin, but the exact location of the one or more endosomal protease cleavage sites within the endogenous activation loop is not limited, as long as the three-dimensional structure of the resulting modified Clostridial neurotoxin is not disrupted and / or the modified Clostridial neurotoxin is not rendered non-functional.

[0078] The entire endogenous activation loop may be replaced with one or more endosomal protease cleavage sites or an exogenous activation loop containing one or more endosomal protease cleavage sites as described herein. Alternatively, a portion or parts of the endogenous activation loop may be replaced (also referred to herein as a partial replacement of the endogenous activation loop), e.g., at least 5, 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the endogenous activation loop are replaced. Preferably, 5 to 20, more preferably 5 to 15, amino acid residues of the endogenous activation loop are replaced. Typically, a partial replacement involves the replacement of consecutive amino acids within the endogenous activation loop. Thus, in a partial replacement of the endogenous activation loop, at least one amino acid residue of the endogenous activation loop is retained. Preferably, about 5 to about 15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residues, for example, about 5 to about 12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) amino acid residues, of the endogenous activation loop are retained. The retained amino acid residues can be at the N-terminus and / or C-terminus of the endogenous activation loop.

[0079] In some embodiments, the endogenous activation loop in the modified clostridial neurotoxins of the present invention is completely replaced. Typically, this means that all amino acid residues in the activation loop (those between the cysteine ​​residues that form the disulfide bond in the active dichain molecule) are replaced with an exogenous activation loop or an exogenous protease cleavage site in accordance with the present invention. Complete replacement of the endogenous activation loop can include introducing an exogenous activation loop consisting entirely of one or more exogenous protease cleavage sites described herein. Alternatively, complete replacement of the endogenous activation loop can include introducing an exogenous activation loop containing one or more exogenous protease cleavage sites described herein along with one or more spacer sequences. Each of the one or more spacer sequences is typically a short peptide (e.g., about 5 to about 25 amino acids, e.g., about 5 to about 20 amino acids, about 5 to about 15 amino acids, or about 5 to about 10 amino acids). Such a spacer may be present when one or more exogenous protease cleavage sites are short motifs (e.g., typically less than 15, preferably less than 10 or 9 amino acids in length). One or more spacers may be present at the N-terminus and / or C-terminus of each of the exogenous protease cleavage sites. Preferably, the spacer may be a GS spacer as defined herein.

[0080] Replacement of the endogenous activation loop can be achieved by any method known in the art. For example, replacement can be achieved by amino acid modification. The endogenous activation loop can be replaced by deleting one or more amino acid residues of the endogenous activation loop. The endogenous activation loop can be replaced by substituting one or more amino acid residues of the endogenous activation loop with amino acid residues of an exogenous activation loop. The endogenous activation loop (or a portion thereof) can be deleted, and one or more endosomal protease cleavage sites, or an exogenous activation loop containing one or more endosomal protease cleavage sites, can be inserted, preferably at the position formally occupied by the endogenous activation loop. Alternatively, the endogenous activation loop can be retained in the modified clostridial neurotoxin of the present invention, or preferably inactivated (e.g., by mutation). It is preferred that the endogenous activation loop (a portion thereof or the entire endogenous activation loop) is not present in the modified clostridial neurotoxin of the present invention. Preferably, one or more endosomal protease cleavage sites, or an exogenous activation loop comprising one or more endosomal protease cleavage sites, occupies the position formally occupied by the endogenous activation loop in a clostridial neurotoxin. For the avoidance of doubt, if an endogenous activation loop is modified to include one or more endosomal protease cleavage sites (e.g., by substituting residues within the endogenous activation loop or by adding one or more amino acids to form one or more endosomal protease cleavage sites within the endogenous activation loop), the modified activation loop is an exogenous activation loop in accordance with the present invention. Thus, potentially, a modified clostridial neurotoxin can contain both its endogenous activation / cleavage site and one or more endosomal protease cleavage sites, and thus can be activated by either the native activating protease (or an equivalent used in recombinant BoNT production, e.g., trypsin or Lys-C) or one or more endosomal proteases.

[0081] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art and can be used to practice the present invention. For example, amino acid modifications can be introduced by modifying the DNA sequence encoding the clostridial neurotoxin. This can be achieved using standard molecular cloning techniques, such as site-directed mutagenesis, in which a short strand of DNA (oligonucleotide) encoding the desired amino acid(s) is used to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting portions of a gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized. Any other method for modifying polypeptides known in the art (e.g., polypeptide synthesis and polypeptide conjugation) can also be used to modify the clostridial neurotoxin according to the present invention.

[0082] An endogenous activation loop replaced in accordance with the present invention may comprise or consist of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, or SEQ ID NO: 112. An endogenous activation loop replaced in accordance with the present invention may comprise or consist of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO:112. In particular, the intrinsic activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, or SEQ ID NO: 112. In particular, the intrinsic activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:112. Preferably, the intrinsic activation loop comprises or consists of the polypeptide sequence set forth as SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, or SEQ ID NO: 112. Preferably, the intrinsic activation loop comprises or consists of the polypeptide sequence set forth as SEQ ID NO:112.

[0083] An endogenous activation loop replaced in accordance with the present invention may comprise or consist of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, or SEQ ID NO:96. An endogenous activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, or SEQ ID NO:96. Preferably, the endogenous activation loop comprises or consists of the polypeptide sequence set forth as SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, or SEQ ID NO:96.

[0084] Preferably, the endogenous activation loop replaced in accordance with the present invention comprises or consists of a polypeptide sequence having at least 70% (e.g., at least 80% or 90%) sequence identity to SEQ ID NO: 94. The endogenous activation loop may comprise or consist of a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 94. More preferably, the endogenous activation loop comprises or consists of the polypeptide sequence set forth as SEQ ID NO: 94.

[0085] The present invention encompasses methods and Clostridial neurotoxins in which the endogenous activation loop is replaced with an exogenous cleavage site that is, or includes, one or more endosomal protease cleavage sites described herein.

[0086] The modified clostridial neurotoxins of the present invention may include an exogenous activation loop containing one or more of any of the endosomal protease cleavage sites described herein. The exogenous activation loop can be produced by replacing one or more amino acids in the endogenous activation loop of a clostridial neurotoxin, as described herein. In some preferred embodiments, the replaced amino acids in the endogenous activation loop are replaced with one or more endosomal protease cleavage sites or exogenous activation loops having the same number of amino acids. In other words, for example, if five amino acids are replaced in the endogenous activation loop, the replaced one or more endosomal protease cleavage sites or the exogenous activation loop containing the one or more endosomal protease cleavage sites will have five amino acids. If ten amino acids are replaced in the endogenous activation loop, the replaced one or more endosomal protease cleavage sites or the exogenous activation loop containing the one or more endosomal protease cleavage sites will have ten amino acids.

[0087] Non-limiting examples of such exogenous activation loops include CQEAANERQQAKKDFFSSHPLREPVNATEDPDLKNVKSGLTNIKTELVTPARDLFGFVGLFRGHHPDC (SEQ ID NO: 127), CQLGKNEEGLFGFVGLFRGHHPDELVTPARDFGHFGLSGLTNIKTEC (SEQ ID NO: 128), and / or CPGGGNKKIELVTPARDLFGFVGLFRGHHPDLKNVKSKC (SEQ ID NO: 129), or corresponding sequences lacking the N- and / or C-terminal cysteine ​​residues (since the remaining sequence can be inserted within the endogenous cysteine ​​residues of the unmodified clostridial neurotoxin, all of which are derived from the BoNT / A1 activation loop).

[0088] The present invention provides a method for producing a modified clostridial neurotoxin according to the present invention, the method comprising replacing an endogenous activation loop (or a portion thereof) of a clostridial neurotoxin with an exogenous activation loop or an exogenous cleavage site to obtain a modified clostridial neurotoxin, wherein the exogenous cleavage site is one or more endosomal protease cleavage sites described herein, or the exogenous activation loop comprises the one or more endosomal protease cleavage sites. Typically, the one or more endosomal protease cleavage sites are selected from a sequence comprising or consisting of the amino acid sequence of SEQ ID NOs: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or 12-34, 130-152, or 171-187, or the exogenous activation loop comprises the one or more endosomal protease cleavage sites.

[0089] The present invention provides modified clostridial neurotoxins (e.g., obtainable by the methods of the present invention), in which the endogenous activation loop (or a portion thereof) of a clostridial neurotoxin is replaced with an exogenous activation loop or an exogenous cleavage site to provide a modified clostridial neurotoxin, wherein the exogenous cleavage site is, or the exogenous activation loop includes, one or more endosomal protease cleavage sites described herein. Typically, the one or more endosomal protease cleavage sites are selected from a sequence comprising or consisting of the amino acid sequence of SEQ ID NOs: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or 12-34, 130-152, or 171-187, or the extrinsic activation loop comprises the one or more endosomal protease cleavage sites (e.g., any one of SEQ ID NOs: 127-129).

[0090] A clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention can be encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, in which the nucleotide sequence encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced by a nucleic acid encoding an extrinsic activation loop comprising one or more endosomal protease sites or the one or more endosomal cleavage sites. A clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention can be encoded by a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 163, in which the nucleotide sequence encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced by a nucleic acid encoding an extrinsic activation loop comprising one or more endosomal protease sites or the one or more endosomal cleavage sites. Preferably, a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention is encoded by the nucleotide sequence of SEQ ID NO: 163, in which the nucleic acid encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced by a nucleotide sequence encoding an extrinsic activation loop containing one or more endosomal protease sites or the one or more endosomal cleavage sites. Non-limiting examples of nucleotide sequences encoding extrinsic activation loops that can replace SEQ ID NO: 164 in SEQ ID NO: 163 include SEQ ID NOs: 165, 166, and 167. Thus, as non-limiting examples, a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention can be encoded by the nucleotide sequence of SEQ ID NOs: 168, 169, and 170. A clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention can comprise a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NOs: 121 or 159-162. A clostridial neurotoxin of the present invention may comprise a polypeptide sequence having at least 80% or 90% sequence identity to one or more of SEQ ID NOs: 121 or 159-162. Preferably, a clostridial neurotoxin of the present invention comprises (more preferably consists of) a polypeptide sequence set forth as any one of SEQ ID NOs: 121 or 159-162.

[0091] The clostridial neurotoxin (e.g., modified clostridial neurotoxin) of the present invention is preferably a retargeted BoNT / X, wherein the clostridial neurotoxin is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site, optionally selected from SEQ ID NOs: 165, 166, and 167. The clostridial neurotoxin may be encoded by a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 163, wherein SEQ ID NO: 164 within SEQ ID NO: 163 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site, optionally selected from SEQ ID NOs: 165, 166, and 167. Preferably, the clostridial neurotoxin is encoded by a nucleotide sequence comprising (or consisting of) SEQ ID NO: 163, in which SEQ ID NO: 164 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site, optionally selected from SEQ ID NOs: 165, 166, and 167. The clostridial neurotoxin of the present invention is preferably a retargeted BoNT / X, which clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 120, in which SEQ ID NO: 91 is replaced by at least one endosomal protease cleavage site or extrinsic activation loop (optionally selected from SEQ ID NOs: 127-129). The clostridial neurotoxin may comprise a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 120, in which SEQ ID NO: 91 is replaced by at least one endosomal protease cleavage site or extrinsic activation loop (optionally selected from SEQ ID NOs: 127-129).The clostridial neurotoxin may comprise a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is replaced by at least one endosomal protease cleavage site or extrinsic activation loop (optionally selected from SEQ ID NOs: 127-129). Preferably, the clostridial neurotoxin comprises (or consists of) the polypeptide sequence set forth as SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is replaced by at least one endosomal protease cleavage site or extrinsic activation loop (optionally selected from SEQ ID NOs: 127-129).

[0092] Polypeptide sequences of the invention (or nucleotide sequences encoding them) may include a purification tag, such as a His tag. The invention is also intended to encompass polypeptide sequences (and nucleotide sequences encoding them) from which the purification tag has been removed.

[0093] Endosomal proteases and endosomal protease cleavage sites Endosomes and lysosomes can contain several different proteases, referred to herein as "endosomal proteases" (also interchangeably referred to as endolysosomal proteases). In other words, the term "endosomal protease," as used herein, refers to a protease that can be contained in an endosome, a lysosome, or both. These endosomal proteases have a variety of different functions and are typically involved in the degradation of proteins and peptides that are imported into endosomes from outside the cell. These enzymes are readily available commercially.

[0094] Non-limiting examples of endosomal proteases according to the present invention include cathepsin and asparagine endopeptidase (AEP, also called asparaginyl endopeptidase or legumain). The term "cathepsin" refers to a family of endosomal proteases, including cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin W, and cathepsin Z. Preferably, the present invention relates to cathepsin L (e.g., L1), B, D, K, or S, and / or AEP. In other words, the modified clostridial neurotoxins of the present invention typically contain at least one cleavage site for one or more of cathepsins L (e.g., L1), B, D, K, or S, and / or AEP.

[0095] Cathepsin L1 is a thiol protease with specificity similar to that of papain, and is involved in the synthesis of Xaa1-Xaa2-Leu / Val / Phe / Ile-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7 (Xaa 1~7 can each be independently selected from any amino acid, and " / / " indicates the position of the peptide bond to be hydrolyzed. The term "cathepsin L1" encompasses the cathepsin L1 described herein as well as any structurally and / or functionally similar (preferably structurally and functionally similar) proteases capable of hydrolyzing the consensus Xaa1-Xaa2-Leu / Val / Phe / Ile-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7 peptide bond. A preferred cathepsin L1 is human cathepsin L1, whose UniProt accession number is P07711 (sequence version 2, deposited October 1, 1989, accessed July 30, 2022) and SEQ ID NO: 39 herein. This sequence is the propeptide, and is converted to the mature human form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 113. Human cathepsin L1 is commercially available from Merck (#SRP6416).

[0096] Cathepsin B is a thiol protease with specificity similar to that of papain, cleaving after the second arginine residue of (i) the consensus sequence Arg-Arg-Xaa (where X is any amino acid) and / or (ii) the consensus sequence Xaa1-Xaa2-Xaa3-Gly / / Xaa4-Xaa5-Gly-Xaa6 (where Xaa 1~6 can each be independently selected from any amino acid, and " / / " indicates the position of the peptide bond to be hydrolyzed. The term "cathepsin B" encompasses the cathepsin B described herein as well as any structurally and / or functionally similar (preferably structurally and functionally similar) protease capable of hydrolyzing the consensus Arg-Arg-Xaa or Xaa1-Xaa2-Xaa3-Gly / / Xaa4-Xaa5-Gly-Xaa6 peptide bond. A preferred cathepsin B is human cathepsin B, whose UniProt accession number is P07858 (sequence version 3, deposited June 21, 2005, accessed July 30, 2022) and is SEQ ID NO: 40 herein. This sequence is the propeptide, and is converted to the mature human form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 79. Human cathepsin B is commercially available from Merck (#SRP0289).

[0097] Cathepsin D is a thiol protease with similar specificity to pepsin A, and is involved in the synthesis of Xaa1-Xaa2-Xaa3-Leu / Phe / / Xaa4-Xaa5-Xaa6-Xaa7 (Xaa 1~7can each be independently selected from any amino acid, and " / / " indicates the position of the peptide bond to be hydrolyzed. The term "cathepsin D" encompasses the cathepsin Ds described herein as well as any structurally and / or functionally similar (preferably structurally and functionally similar) proteases capable of hydrolyzing the consensus Xaa1-Xaa2-Xaa3-Leu / Phe / / Xaa4-Xaa5-Xaa6-Xaa7 peptide bond. A preferred cathepsin D is human cathepsin D, whose UniProt accession number is P07339 (sequence version 1, deposited April 1, 1988, accessed July 30, 2022) and is SEQ ID NO: 41 herein. This sequence is the propeptide, and is converted to the mature human form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 64. Human cathepsin D is commercially available from Merck (#SRP6415).

[0098] Cathepsin K is a thiol protease with a broad range of proteolytic activity. The primary determinant of specificity is P2 (P4-P3-P2-P1 / / P1'-P2'-P3'-P4') in the standard nomenclature, which is preferably Leu, Met, or Phe and not Arg. Cathepsin K is a thiol protease with a broad range of proteolytic activity. 1~7can each be independently selected from any amino acid, and " / / " indicates the position of the peptide bond to be hydrolyzed. The term "cathepsin K" encompasses the cathepsin Ks described herein as well as any structurally and / or functionally similar (preferably structurally and functionally similar) proteases capable of hydrolyzing the consensus Xaa1-Xaa2-Leu / Ile / Val / Pro-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7 peptide bond. A preferred cathepsin K is human cathepsin K, whose UniProt accession number is P43235 (sequence version 1, deposited November 1, 1995, accessed July 30, 2022) and is SEQ ID NO: 42 herein. This sequence is the propeptide, and is converted to the mature human form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 114. Human cathepsin K is commercially available from Merck (#SRP6561).

[0099] Cathepsin S is a thiol protease with a broad range of proteolytic activity. Cathepsin S is a thiol protease with a broad range of proteolytic activity. 1~7 can each be independently selected from any amino acid, and " / / " indicates the position of the peptide bond to be hydrolyzed. The term "cathepsin S" encompasses the cathepsin S described herein as well as any structurally and / or functionally similar (preferably structurally and functionally similar) proteases capable of hydrolyzing the consensus Xaa1-Xaa2-Leu / Val-Xaa3 / / Xaa4-Xaa5-Xaa6-Xaa7 peptide bond. A preferred cathepsin S is human cathepsin S, whose UniProt accession number is P25774 (sequence version 3, deposited February 21, 2006, accessed July 30, 2022) and is SEQ ID NO: 43 herein. This sequence is the propeptide, and is converted to the mature human form by a process involving cleavage of the N-terminal polypeptide from residues 1 to 114. Human cathepsin S is commercially available from Merck (#SRP6297).

[0100] AEP has strict specificity for the hydrolysis of asparaginyl and aspartyl bonds. Thus, AEP cleaves within a series of consensus sequences containing such bonds (Asn / Asp / / Xaa, where Xaa can be any amino acid), which are designated in standard nomenclature as Xaa1-Xaa2-Xaa3-Asn / Asp / / Xaa4-Xaa5-Xaa6-Xaa7 (Xaa 1~7 can each independently be selected from any amino acid and " / / " indicates the position of the peptide bond to be hydrolyzed. The term "AEP" encompasses the AEPs described herein as well as any structurally and / or functionally similar (preferably structurally and functionally similar) proteases capable of hydrolyzing asparaginyl and / or aspartyl bonds. A suitable AEP is human AEP, whose UniProt accession number is Q99538 (sequence version 1, deposited May 1, 1997, accessed July 30, 2022) and is SEQ ID NO: 44 herein. Human AEP is commercially available from Jena Bioscience (#PR-967S or #PR-967L).

[0101] Other exemplary cathepsins include cathepsin A (e.g., UniProt accession number P10619, sequence version 2, deposited April 16, 2002, accessed July 30, 2022), cathepsin C (e.g., UniProt accession number P53634, sequence version 2, deposited January 11, 2011, accessed July 30, 2022), cathepsin E (e.g., UniProt accession number No. P14091, sequence version 3, deposited March 28, 2018, accessed July 30, 2022), cathepsin F (e.g., UniProt accession number Q9UBX1, sequence version 1, deposited May 1, 2000, accessed July 30, 2022), cathepsin G (e.g., UniProt accession number P08311, sequence version 2, deposited January 1, 1990, accessed July 30, 2022), cathepsin H (e.g., UniProt accession number P09668, sequence version 4, deposited February 9, 2010, accessed July 30, 2022), cathepsin O (e.g., UniProt accession number P43234, sequence version 1, deposited November 1, 1995, accessed July 30, 2022), cathepsin V (e.g., UniProt accession number O60911, sequence version Cathepsin W (e.g., UniProt accession number P56202, sequence version 2, deposited on September 22, 2009, accessed on July 30, 2022), and cathepsin Z (e.g., UniProt accession number Q9UBR2, sequence version 1, deposited on May 1, 2000, accessed on July 30, 2022).

[0102] It is within the routine skill of one of ordinary skill in the art to determine the appropriate concentration / unit amount of any endosomal protease to activate the modified Clostridial neurotoxins of the present invention under standard / desired conditions for in vitro and ex vivo use.

[0103] In the context of the present invention, the term "cathepsin L (e.g., L1)" encompasses a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 39, or a mature form thereof. Thus, "cathepsin L (e.g., L1)" can include a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 39, or a mature form thereof. Preferably, cathepsin L (e.g., L1) comprises (more preferably consists of) SEQ ID NO: 39, or a mature form thereof.

[0104] In the context of the present invention, the term "cathepsin B" encompasses a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 40, or a mature form thereof. Thus, "cathepsin B" may include a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 40, or a mature form thereof. Preferably, cathepsin B comprises (more preferably consists of) SEQ ID NO: 40, or a mature form thereof.

[0105] In the context of the present invention, the term "cathepsin D" encompasses a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 41, or a mature form thereof. Thus, "cathepsin D" may include a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 41, or a mature form thereof. Preferably, cathepsin D comprises (more preferably consists of) SEQ ID NO: 41, or a mature form thereof.

[0106] In the context of the present invention, the term "cathepsin K" encompasses a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, or a mature form thereof. Thus, "cathepsin K" may include a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 42, or a mature form thereof. Preferably, cathepsin K comprises (more preferably consists of) SEQ ID NO: 42, or a mature form thereof.

[0107] In the context of the present invention, the term "cathepsin S" encompasses a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 43, or a mature form thereof. Thus, "cathepsin S" may include a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 43, or a mature form thereof. Preferably, cathepsin S comprises (more preferably consists of) SEQ ID NO: 43, or a mature form thereof.

[0108] In the context of the present invention, the term "AEP" encompasses polypeptide sequences having at least 70% sequence identity to SEQ ID NO: 44. Thus, an "AEP" can include polypeptide sequences having at least 80% or 90% sequence identity to SEQ ID NO: 44. Preferably, an AEP comprises (more preferably, consists of) SEQ ID NO: 44.

[0109] In the context of the present invention, the term "cathepsin A" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. P10619 described herein, or a mature form thereof. Thus, "cathepsin A" can include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. P10619 described herein, or a mature form thereof. Preferably, cathepsin A comprises (more preferably consists of) UniProt Accession No. P10619 described herein, or a mature form thereof.

[0110] In the context of the present invention, the term "cathepsin C" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. P53634 described herein, or a mature form thereof. Accordingly, "cathepsin C" may include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. P53634 described herein, or a mature form thereof. Preferably, cathepsin C comprises (more preferably consists of) UniProt Accession No. P53634 described herein, or a mature form thereof.

[0111] In the context of the present invention, the term "cathepsin E" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. P14091 described herein, or a mature form thereof. Accordingly, "cathepsin E" may include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. P14091 described herein, or a mature form thereof. Preferably, cathepsin E comprises (more preferably consists of) UniProt Accession No. P14091 described herein, or a mature form thereof.

[0112] In the context of the present invention, the term "cathepsin F" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. Q9UBX1 described herein, or a mature form thereof. Thus, "cathepsin F" can include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. Q9UBX1 described herein, or a mature form thereof. Preferably, cathepsin F comprises (more preferably consists of) UniProt Accession No. Q9UBX1 described herein, or a mature form thereof.

[0113] In the context of the present invention, the term "cathepsin G" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. P08311 described herein, or a mature form thereof. Thus, "cathepsin G" may include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. P08311 described herein, or a mature form thereof. Preferably, cathepsin G comprises (more preferably consists of) UniProt Accession No. P08311 described herein, or a mature form thereof.

[0114] In the context of the present invention, the term "cathepsin H" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. P09668 described herein, or a mature form thereof. Accordingly, "cathepsin H" may include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. P09668 described herein, or a mature form thereof. Preferably, cathepsin H comprises (more preferably consists of) UniProt Accession No. P09668 described herein, or a mature form thereof.

[0115] In the context of the present invention, the term "cathepsin O" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. P43234 described herein, or a mature form thereof. Accordingly, "cathepsin O" may include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. P43234 described herein, or a mature form thereof. Preferably, cathepsin O comprises (more preferably consists of) UniProt Accession No. P43234 described herein, or a mature form thereof.

[0116] In the context of the present invention, the term "cathepsin V" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. 060911 described herein, or a mature form thereof. Accordingly, "cathepsin V" may include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. 060911 described herein, or a mature form thereof. Preferably, cathepsin V comprises (more preferably consists of) UniProt Accession No. 060911 described herein, or a mature form thereof.

[0117] In the context of the present invention, the term "cathepsin W" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. P56202 described herein, or a mature form thereof. Accordingly, "cathepsin W" may include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. P56202 described herein, or a mature form thereof. Preferably, cathepsin W comprises (more preferably consists of) UniProt Accession No. P56202 described herein, or a mature form thereof.

[0118] In the context of the present invention, the term "cathepsin Z" encompasses a polypeptide sequence having at least 70% sequence identity to UniProt Accession No. Q9UBR2 described herein, or a mature form thereof. Thus, "cathepsin Z" can include a polypeptide sequence having at least 80% or 90% sequence identity to UniProt Accession No. Q9UBR2 described herein, or a mature form thereof. Preferably, cathepsin Z comprises (more preferably consists of) UniProt Accession No. Q9UBR2 described herein, or a mature form thereof.

[0119] In describing the endosomal cleavage sites of the present invention, it is not intended that any Xaa (e.g., Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, or Xaa7) be limited to only one type of amino acid. Thus, one or more residues present at any Xaa can be independently selected from the standard amino acids: aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine.

[0120] Alternatively / additionally, one or more residues present at any Xaa (e.g., Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, or Xaa7) may be independently selected from non-canonical amino acids (amino acids that are not part of the canonical set of 20 amino acids described above). By way of example, non-standard amino acids can include 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and / or ornithine, and 4-fluorophenylalanine. Methods for incorporating non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using E. coli auxotrophic expression hosts.

[0121] The properties of the standard amino acids are shown in the table below. [Table 1]

[0122] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0123] Exemplary (typically consensus) cleavage sites for cathepsins L (e.g., L1), B, D, K, or S, and AEP are described herein. The modified clostridial neurotoxins of the invention may contain one or more of these cleavage sites.

[0124] A cathepsin L (eg, L1) cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0125] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. " / / " indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0126] Therefore, the cathepsin L cleavage site is [ka] In the sequences, the amino acid residues shown in brackets are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. "|" indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0127] The cathepsin B cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0128] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. " / / " indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0129] Therefore, the cathepsin B cleavage site is [ka] In the sequences, the amino acid residues shown in brackets are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. "|" indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0130] The cathepsin D cleavage site may comprise or consist of a consensus sequence selected from the following: [ka]

[0131] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. " / / " indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0132] Therefore, the cathepsin D cleavage site is [ka] In the sequences, the amino acid residues shown in brackets are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. "|" indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0133] The AEP cleavage site may comprise or consist of the following consensus sequence: [ka]

[0134] The amino acid residues shown at each position (P4, P3, P2, P1, P1', P2', P3', and P4') are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. " / / " indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0135] Therefore, the AEP cleavage site is the consensus sequence [ka] In the sequences, the amino acid residues shown in brackets are alternative possible amino acid residues at each position, with the residues shown in bold and underlined being preferred. "|" indicates the cleavage site (i.e., the peptide bond that is hydrolyzed).

[0136] The endosomal protease cleavage sites of the present invention are STSQKSIVAYTMSLGADSS (SEQ ID NO: 12), LFRGGHHPD (SEQ ID NO: 13), ELVTPARDFGHFGLS (SEQ ID NO: 14), QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQA (SEQ ID NO: 15), STSQKSIVAYTMSLGADSSTGFGTNE (SEQ ID NO: 16), LFRGGHHPDTGFGTNE (SEQ ID NO: 17), ELVTPARDFGHFGLSTGFGTNE (SEQ ID NO: 18), QAKKDFFSSHPLREPVN ATEDPSSGYYSTTIRYQATGFGTNE (SEQ ID NO: 19), LFGFVG (SEQ ID NO: 20), ALVEKLLELKKK (SEQ ID NO: 21), QEAANERQQ (SEQ ID NO: 22), SGLTNIKTE (SEQ ID NO: 23), PDLKNVKSK (SEQ ID NO: 24), PGGGNKKIE (SEQ ID NO: 25), QLGKNEEGA (SEQ ID NO: 26), QKVGKAMYAP (SEQ ID NO: 27), GFLG (SEQ ID NO: 28), TVIVITLVMLKKKQ (SEQ ID NO: 29), PVETDSEEQPYLEMDL (SEQ ID NO: 30), LEGME LIVSQVHPETKENEIYPVWSGLP (SEQ ID NO: 31), QKEYALLYKLDIEP (SEQ ID NO: 32), SLAEEEVVIRSED (SEQ ID NO: 33), ERNSNLVGAA (SEQ ID NO: 34), PDLKNVKS (SEQ ID NO: 130), DLFGFVGL (SEQ ID NO: 131), GFVGLFRG (SEQ ID NO: 132), GSGLFGFVGGSG (SEQ ID NO: 133), LFGFVGLFGFVG (SEQ ID NO: 134), LFGFVGLFGFVGLFGFVG (SEQ ID NO: 135), GLFGFVGL (SEQ ID NO: 136), QAKKD FFSSHPLREPVNATED (SEQ ID NO: 137), ELVTPARD (SEQ ID NO: 138), RDFGHFGL (SEQ ID NO: 139), GLFRGHHP (SEQ ID NO: 140), GSGLFRGHHPDGSG (SEQ ID NO: 141), LFRGHHPDLFRGHHPD (SEQ ID NO: 142), ELVTPARDFGHFGLS (SEQ ID NO: 143), QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFLGTNE (SEQ ID NO: 144), LFRGHHPDSTSQKSIVAYTMSLGADSS (SEQ ID NO: 145),STSQKSIVAYTMSLGADSSLFRGHHPD (SEQ ID NO: 146), STSQKSIVAYTMSLGADSSSTSQKSIVAYTMSLGADSS (SEQ ID NO: 147), LFRGHHPDLFRGHHPDLFRGHHPD (SEQ ID NO: 148), ELVTPARDFGHFGLSELVTPARDFGHFGLS (SEQ ID NO: 149), STSQKSIVAYTMSLGADSSELVTPARDFGHFGLSLFRGHHPD (SEQ ID NO: 150), QLGKNEEG (SEQ ID NO: 151), GERGFFYTPKT (SEQ ID NO: 152), GYYSTTIRYQATGFGTNE (SEQ ID NO: 171), GYYSTTIRYQATGFGTNE (SEQ ID NO: 171), LFRGHHPD (SEQ ID NO: 172), GLFRGHHPD (SEQ ID NO: 173), QAKKDFFSS HPLREPVNATEDPSSGYYSTTIRYQATGFGTNEP (SEQ ID NO: 174), QAKKDFFSSHPL (SEQ ID NO: 175), REPVNATEDPSSGYYS (SEQ ID NO: 176), TTIRYQATGFGTNE (SEQ ID NO: 177), TTIRYQATGFGTNEP (SEQ ID NO: 178), EVDLLIGSS (SEQ ID NO: 179), EVDLLIGSSGE (SEQ ID NO: 180), GLAGFLGG (SEQ ID NO: 181), GLFGFVGG (SEQ ID NO: 182), TVGSFGFE (SEQ ID NO: 183), TVGSFGFEGG (SEQ ID NO: 184), LASLLELPEFLLFLQ (SEQ ID NO: 185), GLTTELFSPVD (SEQ ID NO: 186), and / or LERNSNLVGAA (SEQ ID NO: 187).

[0137] Endosomal protease cleavage sites of the present invention may comprise or consist of one or more core cathepsin L cleavage motifs selected from MSLGADSS (SEQ ID NO: 188), LFRGHHP (SEQ ID NO: 189), GLFRGHHP (SEQ ID NO: 190), ELVTPARD (SEQ ID NO: 138), KDFFSSHP (SEQ ID NO: 191), EPVNATED (SEQ ID NO: 192), TGFGTNE (SEQ ID NO: 193), TGFGTNEP (SEQ ID NO: 194), QKVGKAMY (SEQ ID NO: 195), LLIGSS (SEQ ID NO: 196), and / or LLIGSSGE (SEQ ID NO: 197).

[0138] The endosomal protease cleavage sites of the present invention are STSQKSIVAYTMSLGADSS (SEQ ID NO: 12), LFRGGHHPD (SEQ ID NO: 13), ELVTPARDFGHFGLS (SEQ ID NO: 14), QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQA (SEQ ID NO: 15), STSQKSIVAYTMSLGADSS (SEQ ID NO: 16), LFRGGHHPD (SEQ ID NO: 17), ELVTPARDFGHFGLS (SEQ ID NO: 18), QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQAT GFGTNE (SEQ ID NO: 19), QKVGKAMYAP (SEQ ID NO: 27), QAKKDFFSSHPLREPVNATED (SEQ ID NO: 137), ELVTPARD (SEQ ID NO: 138), RDFGHFGL (SEQ ID NO: 139), GLFRGHHP (SEQ ID NO: 140), GSGLFRGHHPDGSG (SEQ ID NO: 141), LFRGHHPDLFRGHHPD (SEQ ID NO: 142), ELVTPARDFGHFGLS (SEQ ID NO: 143), QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFLGTNE (SEQ ID NO: 1 44), LFRGHHPDSTSQKSIVAYTMSLGADSS (SEQ ID NO: 145), STSQKSIVAYTMSLGADSSLFRGHHPD (SEQ ID NO: 146), STSQKSIVAYTMSLGADSSSTSQKSIVAYTMSLGADSS (SEQ ID NO: 147), LFRGHHPDLFRGHHPDLFRGHHPD (SEQ ID NO: 148), ELVTPARDFGHFGLSELVTPARDFGHFGLS (SEQ ID NO: 149), STSQKSIVAYTMSLGADSSELVTPARDFGHFGLSLFRGHHPD ( SEQ ID NO: 150), GYYSTTIRYQATGFGTNE (SEQ ID NO: 171), LFRGHHPD (SEQ ID NO: 172), GLFRGHHPD (SEQ ID NO: 173), QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNEP (SEQ ID NO: 174), QAKKDFFSSHPL (SEQ ID NO: 175), REPVNATEDPSSGYYS (SEQ ID NO: 176), TTIRYQATGFGTNE (SEQ ID NO: 177), TTIRYQATGFGTNEP (SEQ ID NO: 178), EVDLLIGSS (SEQ ID NO: 179),and / or EVDLLIGSSGE (SEQ ID NO: 180).

[0139] Endosomal protease cleavage sites of the present invention may comprise or consist of one or more core cathepsin B cleavage motifs selected from LFGFVF (SEQ ID NO: 20), GLAGFLGG (SEQ ID NO: 181), GLFGFVGG (SEQ ID NO: 182), GSFGFE (SEQ ID NO: 198), and / or GSFGFEGG (SEQ ID NO: 199).

[0140] Endosomal protease cleavage sites of the present invention may comprise or consist of one or more cathepsin B cleavage sites selected from LFGFVG (SEQ ID NO:20), GFLG (SEQ ID NO:28), DLFGFVGL (SEQ ID NO:131), GFVGLFRG (SEQ ID NO:132), GSGLFGFVGGSG (SEQ ID NO:133), LFGFVGLFGFVG (SEQ ID NO:134), LFGFVGLFGFVGLFGFVG (SEQ ID NO:135), GLFGFVGL (SEQ ID NO:136), GLAGFLGG (SEQ ID NO:181), GLFGFVGG (SEQ ID NO:182), TVGSFGFE (SEQ ID NO:183), and / or TVGSFGFEGG (SEQ ID NO:184).

[0141] Endosomal protease cleavage sites of the present invention may comprise or consist of one or more core cathepsin D cleavage motifs selected from VEKLLELK (SEQ ID NO: 200), VITLVMLK (SEQ ID NO: 201), GMELIVSQ (SEQ ID NO: 202), QPYLEMDL (SEQ ID NO: 203), EYALLYKL (SEQ ID NO: 204), LAEEEVVI (SEQ ID NO: 205), LASLLELP (SEQ ID NO: 206), and / or TTELFSPV (SEQ ID NO: 207).

[0142] Endosomal protease cleavage sites of the present invention may comprise or consist of one or more cathepsin D cleavage sites selected from ALVEKLLELKKK (SEQ ID NO: 21), TVIVITLVMLKKKQ (SEQ ID NO: 29), PVETDSEEQPYLEMDL (SEQ ID NO: 30), LEGMELIVSQVHPETKENEIYPVWSGLP (SEQ ID NO: 31), QKEYALLYKLDIEP (SEQ ID NO: 32), SLAEEEVVIRSED (SEQ ID NO: 33), GERGFFYTPKT (SEQ ID NO: 152), LASLLELPEFLLFLQ (SEQ ID NO: 185), and / or GLTTELFSPVD (SEQ ID NO: 186).

[0143] Endosomal protease cleavage sites of the present invention may comprise or consist of one or more core AEP cleavage motifs selected from EAANERQQ (SEQ ID NO: 208), GLTNIKTE (SEQ ID NO: 209), DLKNVKSK (SEQ ID NO: 210), GGGNKKIE (SEQ ID NO: 211), LGKNEEGA (SEQ ID NO: 212), ERNSNLV (SEQ ID NO: 213), and / or LERNSNLV (SEQ ID NO: 214).

[0144] Endosomal protease cleavage sites of the present invention may comprise or consist of one or more AEP cleavage sites selected from QEAANERQQ (SEQ ID NO: 22), SGLTNIKTE (SEQ ID NO: 23), PDLKNVKSK (SEQ ID NO: 24), PGGGNKKIE (SEQ ID NO: 25), QLGKNEEGA (SEQ ID NO: 26), ERNSNLVGAA (SEQ ID NO: 34), PDLKNVKS (SEQ ID NO: 130), QLGKNEEG (SEQ ID NO: 151), and / or LERNSNLVGAA (SEQ ID NO: 187).

[0145] The modified Clostridial neurotoxins of the present invention may contain one or more endosomal protease cleavage sites as defined herein.

[0146] In some embodiments, an endosomal protease cleavage site of the present invention has at least 70% sequence identity to any one of SEQ ID NOs: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or 12-34, 130-152, or 171-187. An endosomal protease cleavage site may have at least 80%, 85%, or 90% sequence identity to any one of SEQ ID NOs: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or 12-34, 130-152, or 171-187. Preferably, the endosomal protease cleavage site has at least 95% sequence identity to any one of SEQ ID NOs: 1 to 38, 130 to 152, or 171 to 187; 12 to 38, 130 to 152, or 171 to 187; 1 to 34, 130 to 152, or 171 to 187; or 12 to 34, 130 to 152, or 171 to 187. More preferably, the endosomal protease cleavage site has at least 99% sequence identity to any one of SEQ ID NOs: 1 to 38, 130 to 152, or 171 to 187; 12 to 38, 130 to 152, or 171 to 187; 1 to 34, 130 to 152, or 171 to 187; or 12 to 34, 130 to 152, or 171 to 187. Particularly preferred are endosomal protease cleavage sites comprising or consisting of any one of SEQ ID NOs: 1 to 38, 130 to 152, or 171 to 187; 12 to 38, 130 to 152, or 171 to 187; 1 to 34, 130 to 152, or 171 to 187; or 12 to 34, 130 to 152, or 171 to 187.

[0147] Typically, the endosomal protease cleavage site comprises or consists of one or more of the amino acid sequences of any one of SEQ ID NOs: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or 12-34, 130-152, or 171-187, or the extrinsic activation loop comprises the one or more endosomal protease cleavage sites.

[0148] As described herein, one or more endosomal protease cleavage sites can be included within the extrinsic activation loop together with one or more spacer sequences as defined herein. A spacer sequence can typically be present when one or more endosomal protease cleavage sites are short motifs (e.g., typically less than 15, preferably less than 10 or less than 9 amino acids in length). One or more spacers can be present at the N-terminus and / or C-terminus of each of the endosomal protease cleavage sites. Preferably, the spacer can be a GS spacer as defined herein.

[0149] The modified clostridial neurotoxins of the present invention may contain one or more endosomal protease cleavage sites. In other words, the modified clostridial neurotoxins of the present invention may contain one endosomal protease cleavage site described herein, or may contain multiple endosomal protease cleavage sites. The modified clostridial neurotoxins of the present invention may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endosomal protease cleavage sites. As a non-limiting example, the modified clostridial neurotoxins of the present invention may contain 2 to 7 (2, 3, 4, 5, 6, or 7) endosomal protease cleavage sites. When the modified clostridial neurotoxins of the present invention contain multiple endosomal protease cleavage sites, each of these may be independently selected. Typically, when the modified clostridial neurotoxins of the present invention contain multiple endosomal protease cleavage sites, each endosomal protease cleavage site may be independently selected from the endosomal protease cleavage sites described herein. Thus, a modified Clostridial neurotoxin of the present invention may contain multiple endosomal protease cleavage sites, each different from the other, a modified Clostridial neurotoxin of the present invention may contain two or more copies of a particular endosomal protease cleavage site, or any combination thereof. Whether an extrinsic activation loop contains multiple different endosomal protease cleavage sites or multiple copies of the same endosomal protease cleavage site, the cleavage sites may be directly linked or separated by one or more spacers as described herein.

[0150] Multiple endosomal protease cleavage sites may be introduced into a single location within a clostridial neurotoxin. Alternatively, multiple endosomal protease cleavage sites may be introduced into multiple locations within a clostridial neurotoxin. As a non-limiting example, one endosomal protease cleavage site may be introduced into the activation loop within a clostridial neurotoxin, and another (the same or different) endosomal protease cleavage site may be introduced into the LH NThe endosomal protease cleavage sites may be introduced into a domain. Typically, when multiple endosomal protease cleavage sites are introduced into a modified clostridial neurotoxin according to the present invention, they are introduced into a single position within the clostridial neurotoxin. Preferably, when multiple endosomal protease cleavage sites are introduced into a modified clostridial neurotoxin according to the present invention, they are each introduced into the activation loop of the clostridial neurotoxin. The relative positioning of individual endosomal protease cleavage sites within the extrinsic activation loop of the present invention can be determined by the structure-function relationship of the endosomal protease in question. Based on this structure-function relationship, it is within the routine skill of a person skilled in the art to appropriately position individual endosomal protease cleavage sites within an extrinsic activation loop containing multiple endosomal protease cleavage sites without excessive burden.

[0151] Examples of polypeptide sequences containing multiple endosomal protease cleavage sites are: [ka]

[0152] The bolded and underlined residue identifies the P1 residue within the endosomal protease cleavage site, i.e., the site after which hydrolysis of the peptide bond occurs. [ka] contains three endosomal protease cleavage sites (two for cathepsin L and one for cathepsin D), [ka] contains two cathepsin L cleavage sites, [ka] contains three endosomal protease cleavage sites (two for cathepsin L and one for cathepsin B), [ka] contains five different AEP cleavage sites.

[0153] Thus, a modified Clostridial neurotoxin of the present invention may comprise one or more of SEQ ID NOs: 35-38.

[0154] The exogenous activation loop containing one or more endosomal protease cleavage sites can be of any length, provided that the structure of the exogenous activation loop, and typically the clostridial neurotoxin, is preserved and cleavage at the one or more endosomal protease activation loop forms the active two-chain form of the modified clostridial neurotoxin. The exogenous activation loop can be about 10 to about 80, e.g., about 10 to about 50, about 10 to about 40, or about 10 to about 30 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) amino acids. The exogenous activation loop can be, for example, about 15 to about 35 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) amino acids long, preferably about 15 to about 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acids long. An exogenous activation loop that is 17 amino acids long is particularly preferred, since this is the same length as the endogenous BoNT / C (BoNT / C1) activation loop.

[0155] Clostridial neurotoxins The term "neurotoxin," as used herein, refers to any polypeptide that enters neurons and inhibits neurotransmitter release. This process involves binding of the neurotoxin to low- or high-affinity receptors, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin to the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "neurotoxin" encompasses any polypeptide produced by Clostridial bacteria (clostridial neurotoxins) that enters neurons and inhibits neurotransmitter release, as well as those produced by recombinant or chemical techniques. This two-chain form is the active form of the toxin. The two chains are referred to as the heavy chain (H chain) (molecular weight approximately 100 kDa) and the light chain (L chain) (molecular weight approximately 50 kDa).

[0156] The clostridial neurotoxins of the present invention may be catalytically active (also referred to as active) or catalytically inactive. Preferably, the clostridial neurotoxins of the present invention are catalytically active.

[0157] The terms "catalytically active" and "active" are used interchangeably herein and refer to a clostridial neurotoxin L chain (or a clostridial neurotoxin containing such an L chain) that has non-cytotoxic protease activity. Specifically, an active clostridial neurotoxin L chain has endopeptidase activity and is capable of cleaving a protein of the extracellular fusion apparatus within a target cell. The protein of the extracellular fusion apparatus is preferably a SNARE protein (e.g., SNAP25, synaptobrevin / VAMP, or syntaxin).

[0158] The term "catalytically inactive," as used herein with respect to a clostridial neurotoxin L chain, means that the L chain exhibits substantially no non-cytotoxic protease activity; preferably, the term "catalytically inactive," as used herein with respect to a clostridial neurotoxin L chain, means that the L chain exhibits no non-cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin L chain does not cleave proteins of the exocellular fusion apparatus within a target cell. The term "substantially no non-cytotoxic protease activity" means that a clostridial neurotoxin L chain has less than 5% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain (e.g., less than 2%, less than 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 with the amount of SNARE protein cleaved by a catalytically active clostridial neurotoxin L chain under the same conditions. Routine techniques (e.g., SDS-PAGE and Western blotting) can be used to quantify the amount of cleaved SNARE protein. A suitable in vitro assay is described in WO2019 / 145577A1 (incorporated herein by reference).

[0159] The Clostridial neurotoxin (e.g., before modification) can be BoNT / A. An exemplary reference BoNT / A sequence is the BoNT / A1 sequence set forth as SEQ ID NO: 45 or 117. Other non-limiting examples of BoNT / A sequences include the sequences of SEQ ID NOs: 46-52.

[0160] The Clostridial neurotoxin (e.g., before modification) can be BoNT / B. An exemplary reference BoNT / B sequence is the BoNT / B1 sequence shown as SEQ ID NO: 53. Other non-limiting examples of BoNT / B sequences include the sequences of SEQ ID NOs: 54-60.

[0161] The Clostridial neurotoxin (e.g., before modification) can be BoNT / C. An exemplary reference BoNT / C1 sequence is shown as SEQ ID NO:61.

[0162] The Clostridial neurotoxin (e.g., before modification) can be BoNT / D. An exemplary reference BoNT / D sequence is shown as SEQ ID NO:62.

[0163] The clostridial neurotoxin (e.g., before modification) can be a BoNT / CD chimera. An exemplary reference BoNT / CD sequence is shown as SEQ ID NO:63.

[0164] The Clostridial neurotoxin (e.g., before modification) can be a BoNT / DC chimera. An exemplary reference BoNT / DC sequence is shown as SEQ ID NO:64.

[0165] The Clostridial neurotoxin (e.g., before modification) can be BoNT / E. An exemplary reference BoNT / E sequence is shown as SEQ ID NO: 65. Other non-limiting examples of BoNT / E sequences include the sequences of SEQ ID NOs: 66-77.

[0166] The Clostridial neurotoxin (e.g., before modification) can be BoNT / F. An exemplary reference BoNT / F sequence is the BoNT / F1 sequence shown as SEQ ID NO: 78. Other non-limiting examples of BoNT / F sequences include the sequences of SEQ ID NOs: 79-84.

[0167] The Clostridial neurotoxin (e.g., before modification) can be BoNT / G. An exemplary reference BoNT / G sequence is shown as SEQ ID NO:85.

[0168] The Clostridial neurotoxin (e.g., before modification) can be a BoNT / FA chimera. An exemplary reference BoNT / FA sequence is shown as SEQ ID NO:86.

[0169] The Clostridial neurotoxin (e.g., before modification) can be BoNT / X. An exemplary reference BoNT / X sequence is shown as SEQ ID NO:87.

[0170] The Clostridial neurotoxin (e.g., before modification) can be TeNT. An exemplary reference TeNT sequence is shown as SEQ ID NO:88.

[0171] In some preferred embodiments, the clostridial neurotoxin (e.g., before modification) is BoNT / A, BoNT / B, or BoNT / X, as described herein, or a chimera thereof (e.g., BoNT / AB).

[0172] As explained above, activated clostridial neurotoxins are formed from two polypeptide chains, a heavy chain (H chain) (molecular weight approximately 100 kDa) and a light chain (L chain) (molecular weight approximately 50 kDa). The H chain contains a C-terminal targeting component (receptor binding domain or H chain). C domain) and the N-terminal translocation component (H N domain).

[0173] Examples of light chain reference sequences include the following: Botulinum neurotoxin type A: amino acid residues 1-448 Botulinum neurotoxin type B: amino acid residues 1-440 Botulinum neurotoxin type C1: amino acid residues 1-441 Botulinum neurotoxin type D: amino acid residues 1-445 Botulinum neurotoxin type E: amino acid residues 1-422 Botulinum neurotoxin type F: amino acid residues 1-439 Botulinum neurotoxin type G: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457

[0174] For the recently identified BoNT / X, the L chain is reported to correspond to amino acids 1 to 439, and the boundary of the L chain may vary by approximately 25 amino acids (e.g., 1 to 414 or 1 to 464). Preferably, the L chain in the modified clostridial neurotoxin of the present invention is the BoNT / X chain.

[0175] The reference sequences identified above should be considered as a guideline, as slight variations may occur depending on the subserotype. For example, US2007 / 0166332 (incorporated herein by reference in its entirety) cites slightly different Clostridial sequences. Botulinum neurotoxin type A: amino acid residues M1 to K448 Botulinum neurotoxin type B: amino acid residues M1 to K441 Botulinum neurotoxin type C1: amino acid residues M1 to K449 Botulinum neurotoxin type D: amino acid residues M1 to R445 Botulinum neurotoxin type E: amino acid residues M1 to R422 Botulinum neurotoxin type F: amino acid residues M1 to K439 Botulinum neurotoxin type G: amino acid residues M1 to K446 Tetanus neurotoxin: amino acid residues M1 to A457

[0176] Alternatively, a clostridial neurotoxin L chain can be defined as the amino acid sequence from the first amino acid (including or excluding the first methionine residue) to the first cysteine ​​residue of the intrinsic activation loop. Additionally or alternatively, a clostridial neurotoxin L chain can be defined as the amino acid sequence N-terminal to the cleavage site within the intrinsic activation loop.

[0177] A clostridial neurotoxin L chain can be defined as a clostridial neurotoxin domain that contains a metal-coordinating HExxH motif (SEQ ID NO: 113), which typically functions to cleave SNARE protein substrates.

[0178] The term "light chain" (or "L chain") encompasses variants and fragments thereof, provided that the variants and fragments still exhibit non-cytotoxic protease activity (which can be determined using standard assays known in the art, examples of which are described herein). By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology to a reference L chain. The term "fragment", when used in reference to an L chain, refers to a peptide having at least 200, preferably at least 250, more preferably at least 300, even more preferably at least 350, and most preferably at least 400 amino acid residues of the reference L chain. In the case of a clostridial L chain, the fragment is preferably at least 300, more preferably at least 350, and most preferably at least 400 amino acid residues of the reference L chain. An L chain "fragment" of the present invention encompasses a fragment of a variant L chain based on a reference sequence.

[0179] A clostridial neurotoxin H chain can be defined as the sequence from the second cysteine ​​to the last amino acid of the intrinsic activation loop. Additionally or alternatively, a clostridial neurotoxin H chain can be defined as beginning with an amino acid sequence C-terminal to the cleavage site in the intrinsic activation loop. Additionally or alternatively, a clostridial neurotoxin H chain can be defined as beginning with an amino acid C-terminal to the cysteine ​​residue (typically the second cysteine ​​residue) that forms a disulfide bond between the L chain and the H chain and thus defines the C-terminus of the intrinsic activation loop.

[0180] A translocation domain is a molecule that allows a protease to translocate into a target cell such that functional expression of protease activity occurs in the cytoplasm of the target cell. Whether any molecule (e.g., a protein or peptide) has the requisite translocation function of the present invention can be confirmed by any one of several conventional assays.

[0181] For example, Shone C. (1987) describes an in vitro assay using liposomes exposed to test molecules. The presence of the essential translocation function is confirmed by K + and / or the release of labeled NAD from the liposomes, which can be easily monitored (Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180).

[0182] A further example is provided by Blaustein R. (1987), who describes a simple in vitro assay using planar phospholipid bilayer membranes, which are exposed to a test molecule and the essential translocation function is confirmed by an increase in conductance across the membrane (see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120).

[0183] Further methodology for assessing membrane fusion and enabling the identification of translocation domains suitable for use in the present invention is provided in Methods in Enzymology Vol. 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

[0184] The present invention also encompasses variants and / or fragments of translocation domains, so long as the variant domain still exhibits the requisite translocation activity. By way of example, a variant may have at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% or at least 98% amino acid sequence homology to the reference translocation domain. The term fragment, when used in reference to a translocation domain, refers to a peptide having at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of the reference translocation domain. In the case of a clostridial translocation domain, the fragment preferably has at least 20 amino acid residues of the reference translocation domain (e.g., H N A translocation "fragment" of the present invention includes a fragment of a variant translocation domain based on a reference sequence.

[0185] The Translocation Domain is preferably capable of forming ion-permeable pores in lipid membranes under low pH conditions, and preferably, this is known to use only the portion of the protein molecule capable of pore formation within the endosomal membrane.

[0186] The Translocation Domain may be derived from a microbial protein source, particularly a bacterial or viral protein source. Thus, the Translocation Domain may be the Translocation Domain of an enzyme, such as a bacterial toxin or a viral protein.

[0187] It has been well documented that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains can be used in the present invention.

[0188] The translocation domain may be of clostridial origin (e.g., H N domain (or functional component thereof). N means a portion or fragment of the H chain of a clostridial neurotoxin equivalent to approximately the amino-terminal half of the H chain, or the domain corresponding to that fragment within the intact H chain. C The function is H C It is removed by deletion of amino acid sequences (either at the level of DNA synthesis or at the post-synthetic level by treatment with nucleases or proteases). C The function may be inactivated by chemical or biological treatment, and therefore the heavy chain may be unable to bind to the binding site on the target cell to which the native clostridial neurotoxin (i.e., the holotoxin) binds.

[0189] Examples of suitable (reference) translocation domains include: Botulinum neurotoxin type A: amino acid residues (449-871) Botulinum neurotoxin type B: amino acid residues (441-858) Botulinum neurotoxin type C: amino acid residues (442-866) Botulinum neurotoxin type D: amino acid residues (446-862) Botulinum neurotoxin type E: amino acid residues (423-845) Botulinum neurotoxin type F: amino acid residues (440-864) Botulinum neurotoxin type G: amino acid residues (442-863) Botulinum neurotoxin type X: amino acid residues (461-890) Tetanus neurotoxin: amino acid residues (458-879)

[0190] The translocation domain of the recently identified BoNT / X has been reported to correspond to amino acids 460-890. C The boundary can vary by approximately 10 amino acids, for example, 461 to 889 or 454 to 891. Preferably, the translocation domain of a modified clostridial neurotoxin of the invention is a BoNT / X translocation domain.

[0191] The reference sequences identified above should be considered as a guideline, as slight variations may occur depending on the subserotype. For example, US2007 / 0166332 (incorporated herein by reference) cites slightly different Clostridial sequences. Botulinum neurotoxin type A: amino acid residues (A449-K871) Botulinum neurotoxin type B: amino acid residues (A442-S858) Botulinum neurotoxin type C: amino acid residues (T450-N866) Botulinum neurotoxin type D: amino acid residues (D446-N862) Botulinum neurotoxin type E: amino acid residues (K423-K845) Botulinum neurotoxin type F: amino acid residues (A440 to K864) Botulinum neurotoxin type G: amino acid residues (S447-S863) Tetanus neurotoxin: amino acid residues (S458-V879)

[0192] In the context of the present invention, various clostridial neurotoxins H containing a translocation domain N Regions may be useful in embodiments of the present invention insofar as these active fragments facilitate the release of non-cytotoxic proteases (e.g., clostridial light chains) from intracellular vesicles into the cytoplasm of target cells, allowing clostridial neurotoxins to participate in the overall cellular machinery by which they proteolytically cleave substrates. NThe region is approximately 410-430 amino acids long and contains the translocation domain. N It has been shown that the total length of the region is not required for the translocation activity of the Translocation Domain. Thus, in the context of the present invention, a Translocation Domain refers to a Clostridial neurotoxin H containing a Translocation Domain of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids in length. N Also included are, for example, Clostridial neurotoxin H containing a translocation domain of up to 350 amino acids, up to 375 amino acids, up to 400 amino acids, and up to 425 amino acids in length. N Areas are also included.

[0193] For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al. (1997) (The Clostridia: Molecular Biology and Pathogenesis, Academic press).

[0194] H N The term refers to the naturally occurring neurotoxin H N a modified H having a non-naturally occurring amino acid sequence; N and / or synthetic amino acid residues (except modified H N (Only if the part still exhibits the translocation function described above).

[0195] Alternatively, the translocation domain may be of non-clostridial origin. Examples of non-clostridial (see references) translocation domain origins include, but are not limited to, the diphtheria toxin translocation domain (O'Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51), the Pseudomonas exotoxin type A translocation domain (Prior et al. Biochemistry (1992) 31, 3555-3559), the anthrax toxin translocation domain (Blanke et al. al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442), various fusogenic or hydrophobic peptides with translocation function (Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al. (1992) PNAS, 89, pp. 7934-7938), and amphipathic peptides (Murata et al. (1992) Biochem., 31, pp. 1986-1992). Translocation domains may reflect those present in naturally occurring proteins or may contain amino acid mutations as long as they do not destroy the translocation ability of the translocation domain.

[0196] Specific examples of viral (reference) translocation domains suitable for use in the present invention include certain translocation domains of virally expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e., membrane fusion and vesiculation) functions of several fusogenic and amphipathic peptides derived from the N-terminal region of influenza virus hemagglutinin. Other virally expressed membrane fusion proteins known to have desirable translocation activity include the translocation domain of the fusogenic peptide of Semliki Forest virus (SFV), the translocation domain of vesicular stomatitis virus (VSV) glycoprotein G, the translocation domain of the SER virus F protein, and the translocation domain of the foamy virus envelope glycoprotein. Virus-encoded spike proteins have particular utility in the context of the present invention (e.g., the E1 protein of SFV and the G protein of VSV).

[0197] Use of the translocation domains listed in the table (see below) includes use of sequence variants thereof. Variants can include one or more conservative nucleic acid substitutions and / or nucleic acid deletions or insertions, so long as they retain the essential translocation function. Variants can also include one or more amino acid substitutions and / or amino acid deletions, insertions, or indels, so long as they retain the essential translocation function. [Table 2]

[0198] Clostridial neurotoxin H C Examples of domain reference sequences include: BoNT / A:N872-L1296 BoNT / B:E859-E1291 BoNT / C1:N867-E1291 BoNT / D:S863-E1276 BoNT / E:R846-K1252 BoNT / F:K865-E1274 BoNT / G:N864-E1297 TeNT:I880-D1315

[0199] Regarding the recently identified BoNT / X, C The domain is reported to correspond to amino acids 893 to 1306, and the domain boundary may vary by approximately 25 amino acids (e.g., 868 to 1306 or 918 to 1306). Preferably, the H of the modified clostridial neurotoxin of the present invention C The domain is BoNT / XH C It is a domain.

[0200] The clostridial neurotoxins described herein can further comprise a translocation-facilitating domain, which facilitates delivery of a non-cytotoxic protease into the cytoplasm of a target cell, as described, for example, in WO08 / 008803 and WO08 / 008805, each of which is incorporated herein by reference.

[0201] For example, suitable translocation-facilitating domains include enveloped virus fusogenic peptide domains, e.g., influenza virus fusogenic peptide domains (e.g., a 23-amino acid influenza A virus fusogenic peptide domain), alphavirus fusogenic peptide domains (e.g., a 26-amino acid Semliki Forest virus fusogenic peptide domain), vesiculovirus fusogenic peptide domains (e.g., a 21-amino acid vesicular stomatitis virus fusogenic peptide domain), respirovirus fusogenic peptide domains (e.g., a 25-amino acid Sendai virus fusogenic peptide domain), Examples of such a peptide domain include a morbillivirus fusogenic peptide domain (e.g., a 25-amino acid canine distemper virus fusogenic peptide domain), an avulavirus fusogenic peptide domain (e.g., a 25-amino acid Newcastle disease virus fusogenic peptide domain), a henipavirus fusogenic peptide domain (e.g., a 25-amino acid Hendra virus fusogenic peptide domain), a metapneumovirus fusogenic peptide domain (e.g., a 25-amino acid human metapneumovirus fusogenic peptide domain), or a spumavirus fusogenic peptide domain, such as a simian foamy virus fusogenic peptide domain, or a fragment or variant thereof.

[0202] As a further example, the translocation-facilitating domain may be a domain of Clostridial neurotoxin H CN The clostridial neurotoxin H domain may be a fragment or variant thereof. CN The translocation-facilitating domain may be at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, or at least 275 amino acids in length. CN The length of the translocation facilitating domain is preferably 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: Botulinum neurotoxin type A: amino acid residues (872-1110) Botulinum neurotoxin type B: amino acid residues (859-1097) Botulinum neurotoxin type C: amino acid residues (867-1111) Botulinum neurotoxin type D: amino acid residues (863-1098) Botulinum neurotoxin type E: amino acid residues (846-1085) Botulinum neurotoxin type F: amino acid residues (865-1105) Botulinum neurotoxin type G: amino acid residues (864-1105) Botulinum neurotoxin type X: amino acid residues (890-1121) Tetanus neurotoxin: amino acid residues (880-1127)

[0203] The above sequence positions may vary slightly depending on the serotype / subtype, and the appropriate (reference) Clostridial neurotoxin H CN Further examples of domains include: Botulinum neurotoxin type A: amino acid residues (874-1110) Botulinum neurotoxin type B: amino acid residues (861-1097) Botulinum neurotoxin type C: amino acid residues (869-1111) Botulinum neurotoxin type D: amino acid residues (865-1098) Botulinum neurotoxin type E: amino acid residues (848-1085) Botulinum neurotoxin type F: amino acid residues (867-1105) Botulinum neurotoxin type G: amino acid residues (866-1105) Tetanus neurotoxin: amino acid residues (882-1127)

[0204] Any of the above-described facilitating domains can be combined with any of the previously described translocation domain peptides suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain can be combined with a non-clostridial translocation domain peptide, or with a clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin H CN The translocation facilitating domain may be combined with a non-clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin H CN The facilitating domain may be combined with a Clostridial translocation domain peptide, examples of which include: Botulinum neurotoxin type A: amino acid residues (449-1110) Botulinum neurotoxin type B: amino acid residues (442-1097) Botulinum neurotoxin type C: amino acid residues (450-1111) Botulinum neurotoxin type D: amino acid residues (446-1098) Botulinum neurotoxin type E: amino acid residues (423-1085) Botulinum neurotoxin type F: amino acid residues (440-1105) Botulinum neurotoxin type G: amino acid residues (447-1105) Tetanus neurotoxin: amino acid residues (458-1127)

[0205] In some embodiments, the clostridial neurotoxin of the present invention comprises a functional H C Thus, the clostridial neurotoxin may lack a specific domain in a binding assay (e.g., Clostridial H CThe clostridial neurotoxin is unable to bind to rat synaptosomal membranes (via a nucleotide component) (as described by Shone et al. (1985) Eur. J. Biochem. 151, 75-82). The clostridial neurotoxin may preferably lack the last 50 C-terminal amino acids of the clostridial neurotoxin holotoxin. The clostridial neurotoxin may 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 acids of the clostridial neurotoxin holotoxin. Alternatively, H C The binding activity can be reduced / decreased by mutagenesis. For convenience, referring to BoNT / A, for example, modification of one or two amino acid residue mutations in the ganglioside binding pocket (W1266→L and Y1267→F) can reduce / reduce the binding activity of H C Similar mutations may be made to non-serogroup A clostridial peptide components (e.g., constructs based on botulinum B (W1262→L and Y1263→F) or botulinum E (W1224→L and Y1225→F) with the mutations). Other mutations to the active site may result in the loss of receptor binding function of the same H C Loss of receptor binding activity is achieved (e.g., Y1267S in botulinum toxin type A and the corresponding highly conserved residue in other clostridial neurotoxins). Details of this and other mutations are described in Rummel et al. (2004) (Molecular Microbiol. 51:631-634), which is incorporated herein by reference.

[0206] Native clostridial neurotoxin H C The peptide contains approximately 400-440 amino acid residues and consists of two functionally distinct domains (each approximately 25 kDa): the N-terminal region (generally H CN peptide or domain) and the C-terminal region (generally H CCThis fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: 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; A(2004) Mol. Microbiol. 51(3), 631-643. Furthermore, the C-terminal region (H CC It is well established, and the above publications support, that the C-terminal 160-200 amino acid residues of the heavy chain H are involved in the binding of clostridial neurotoxins to their natural cellular receptors (i.e., nerve endings at the neuromuscular junction). Therefore, throughout this specification, the term "functional heavy chain H" is used. C When referring to a clostridial heavy chain that lacks a peptide (or domain) and is therefore unable to bind to the cell surface receptor to which native clostridial neurotoxins bind, this is meant to imply that the clostridial heavy chain is merely a functional H CC This means that the peptide is absent. CC The peptide region can be partially or entirely deleted or otherwise modified (e.g., by conventional chemical or proteolytic treatment) to inactivate its native binding ability to nerve endings at the neuromuscular junction.

[0207] Therefore, the Clostridial neurotoxin H of the present invention N The peptide may be extended at the C-terminus, i.e., e.g., Clostridial neurotoxin H CAll or part of a domain (e.g., H CN , H CC , or H C As used herein, the clostridial neurotoxin H of the present invention may be bound to the clostridial neurotoxin H N When referring to peptides, such C-terminally extended H N peptide, which is Clostridial neurotoxin H C Alternatively, the clostridial neurotoxin H of the present invention comprises one or more amino acid residues from the N The peptide binds to Clostridial neurotoxin H C All or part of a domain (e.g., H CN , or H CC , or H C domain) may not be bound to (or may be absent from) the

[0208] Typically, the clostridial neurotoxin or clostridial neurotoxin H of the present invention N The peptides consist of the C-terminal peptide portion (H CC ) is absent in whole or in part, the native clostridial neurotoxin H C The binding function is lacking. For example, the C-terminally extended Clostridium H N The peptide may lack 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 the Clostridial neurotoxin heavy chain. N The peptides consist of the C-terminal peptide portion (H CC ) may be missing entirely, thus resulting in the native Clostridial neurotoxin H C It lacks binding function. For example, Clostridium H NThe peptide may lack 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 the clostridial neurotoxin heavy chain. N The peptide is a Clostridium H selected from the group consisting of: CC Reference sequences are missing. Botulinum neurotoxin type A: amino acid residues (Y1111 to L1296) Botulinum neurotoxin type B: amino acid residues (Y1098-E1291) Botulinum neurotoxin type C: amino acid residues (Y1112 to E1291) Botulinum neurotoxin type D: amino acid residues (Y1099-E1276) Botulinum neurotoxin type E: amino acid residues (Y1086-K1252) Botulinum neurotoxin type F: amino acid residues (Y1106 to E1274) Botulinum neurotoxin type G: amino acid residues (Y1106-E1297) Botulinum neurotoxin type X: amino acid residues (Y1122-D1306) Tetanus neurotoxin amino acid residues (Y1128-D1315)

[0209] The reference sequences identified above should be considered as a guideline, as slight variations may occur depending on the subserotype.

[0210] The present invention is suitable for application to a wide variety of clostridial neurotoxins. Thus, in the context of the present invention, the term "clostridial neurotoxin" includes toxins produced by the following bacteria: C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, 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. The term "clostridial neurotoxin" also includes botulinum neurotoxin serotype H. In some preferred embodiments, the clostridial neurotoxin is BoNT / A, more preferably BoNT / A1. In other preferred embodiments, the clostridial neurotoxin is BoNT / X.

[0211] Botulinum neurotoxins (BoNTs) are produced by C. botulinum in the form of large protein complexes consisting of BoNT itself and complexes with several accessory proteins. Currently, there are nine different classes of botulinum neurotoxins, namely, botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and such serotype classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further classified into different subtypes based on the percentage of amino acid sequence identity.

[0212] BoNTs are absorbed in the gastrointestinal tract and enter the systemic circulation, where they bind to the presynaptic membranes of cholinergic nerve terminals and prevent their release of the 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 25 kDa synaptosomal-associated protein (SNAP-25). BoNT / C1 cleaves syntaxin. BoNT / X has been shown to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

[0213] Tetanus toxin is produced by C. tetani in a single serotype: C. butyricum produces BoNT / E, while C. baratii produces BoNT / F.

[0214] The term "clostridial neurotoxin" is intended to encompass modified clostridial neurotoxins and their derivatives, including, but not limited to, those described below. A modified clostridial neurotoxin or derivative can contain one or more amino acids that are modified compared to the native (unmodified) form of the clostridial neurotoxin, or can contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. For example, a modified clostridial neurotoxin can have a modified amino acid sequence in one or more domains relative to the sequence of a native (unmodified) clostridial neurotoxin. Such modifications may alter functional aspects of the toxin (e.g., biological activity and persistence). Thus, the clostridial neurotoxin of the present invention can be a modified clostridial neurotoxin, a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative. Specifically, the modified clostridial neurotoxin of the present invention can be a modified modified clostridial neurotoxin, a modified modified clostridial neurotoxin derivative, or a modified clostridial neurotoxin derivative.

[0215] Modified clostridial neurotoxins contain one or more modifications within the amino acid sequence of the heavy chain (e.g., modified H C domain), and the modified heavy chain binds to target neurons with greater or less affinity than the native (unmodified) Clostridial neurotoxin. C Such modifications in the domain are C This may involve modifying residues within the ganglioside binding site of the domain, or within the protein (SV2 or synaptotagmin) binding site that alters binding to ganglioside and / or protein receptors on target neurons. Examples of such modified Clostridial neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entireties.

[0216] Most preferably, BoNT / BH C The H domain has the effect of increasing the binding affinity of BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence. CC Further comprising a substitution, insertion, indel, or deletion of at least one amino acid residue in the subdomain. CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains are disclosed in WO2013 / 180799 and WO2016 / 154534 (both of which are incorporated herein by reference).

[0217] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains may include substitution mutations selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

[0218] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomain may further comprise a combination of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0219] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains may also include a combination of the three substitution mutations (E1191M, S1199W, and W1178Q).

[0220] Preferably, BoNT / BH CC The substitution, insertion, indel, or deletion of an amino acid residue in the subdomain comprises a combination of two substitution mutations (E1191M and S1199Y). Such modifications are present in the chimeric clostridial neurotoxin of SEQ ID NO: 118. E1191M may correspond to position 1204 of SEQ ID NO: 118, and S1199Y may correspond to position 1212. Thus, SEQ ID NO: 118 may comprise 1204M and 1212Y.

[0221] The modifications may be relative to the unmodified BoNT / B set forth as SEQ ID NO: 53, and the amino acid residue numbering is determined by alignment with SEQ ID NO: 53. Because the presence of a methionine residue at position 1 of SEQ ID NO: 53 (and SEQ ID NOs corresponding to other clostridial neurotoxin polypeptides described herein, including chimeric clostridial neurotoxin polypeptides) is optional, one skilled in the art would take the presence / absence of the methionine residue into consideration when determining the amino acid residue numbering. For example, if SEQ ID NO: 53 contains a methionine, the numbering of the positions will be as defined above (e.g., E1191 becomes E1191 in SEQ ID NO: 53). Alternatively, if a methionine is not present in SEQ ID NO: 53, the numbering of the amino acid residues will be changed by -1 (e.g., E1191 becomes E1190 in SEQ ID NO: 53). Thus, the first methionine amino acid residue in the polypeptide sequence of a chimeric clostridial neurotoxin may be optional or absent. Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and one of skill in the art will readily determine the correct amino acid residue numbering using techniques routine in the art. Alignments can be performed using any of the methods described herein to determine sequence homology and / or percent sequence identity.

[0222] The modified clostridial neurotoxin can have one or more modifications in the amino acid sequence of the light chain (e.g., modifications in the substrate binding domain or catalytic domain that can alter or modify the specificity of the modified light chain for a SNARE protein). Examples of such modified clostridial neurotoxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated by reference in their entireties.

[0223] The modified clostridial neurotoxin may contain one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified clostridial neurotoxin. For example, the modified clostridial neurotoxin may contain a leucine- or tyrosine-based motif, which increases or decreases the biological activity and / or biological persistence of the modified clostridial neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM (where x is any amino acid). Suitable tyrosine-based motifs include Yxx-Hy (where Hy is a hydrophobic amino acid). Examples of modified clostridial neurotoxins containing leucine- and tyrosine-based motifs are described in WO2002 / 008268, which is incorporated herein by reference in its entirety.

[0224] The term "clostridial neurotoxin" is intended to encompass hybrid and chimeric clostridial neurotoxins. A hybrid clostridial neurotoxin comprises at least a portion of the light chain of one clostridial neurotoxin or subtype and at least a portion of the heavy chain of another clostridial neurotoxin or clostridial neurotoxin subtype. A hybrid clostridial neurotoxin may comprise the entire light chain of one clostridial neurotoxin subtype and the heavy chain of another clostridial neurotoxin subtype. A chimeric clostridial neurotoxin may comprise a portion of the heavy chain (e.g., the binding domain) of one clostridial neurotoxin subtype, with the other portion of the heavy chain being from another clostridial neurotoxin subtype. Chimeric clostridial neurotoxins, particularly chimeric BoNTs, comprise four major domains of the neurotoxin: L chain, H chain, and H chain. N , H CN , and H CC The LH (before modification) of SEQ ID NO: 118 may be defined in terms of a serotype or subserotype (as defined herein). N / A1-H CThe B1 chimera may be described as an AABB chimera. Similarly, or alternatively, a therapeutic element may comprise light chain portions of different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of providing the therapeutic benefit of a given clostridial neurotoxin to patients who are immunologically tolerant to a given clostridial neurotoxin subtype, who may have lower-than-average receptor concentrations for a given clostridial neurotoxin heavy chain binding domain, or who may have protease-resistant variants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in U.S. Pat. No. 8,071,110, the contents of which are incorporated herein by reference in their entirety. Thus, the clostridial neurotoxins of the present invention may be hybrid or chimeric clostridial neurotoxins. Specifically, the modified clostridial neurotoxins of the present invention may be modified hybrid or chimeric clostridial neurotoxins.

[0225] In some preferred embodiments, the clostridial neurotoxin is a BoNT / A (e.g., a BoNT / A hybrid or chimera) that includes at least one domain from a non-BoNT / A clostridial neurotoxin. For example, a clostridial neurotoxin of the present invention (one that includes one or more endosomal protease cleavage sites) can include: i. BoNT / AL chain and non-BoNT / AH N and H C domain, ii.BoNT / AH N Domains and non-BoNT / AL chains and H C domain, iii. BoNT / AH C Domains and non-BoNT / AL chains and H N domain, iv.BoNT / AL chain and H N Domain and non-BoNT / AH C domain, v.BoNT / AL chain and H C Domain and non-BoNT / AH N domain, or vi.BoNT / AH N Domain and H C domains and non-BoNT / AL chains.

[0226] As a non-limiting example, a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention may comprise a BoNT / AL chain and a H N Domains and BoNT / BH C domain (e.g., LH N / A1-H C / B1). Exemplary unmodified LHs that can be modified to contain one or more endosomal protease cleavage sites according to the present invention. N / A1-H C The B1 chimera is shown in SEQ ID NO: 118. N / A1-H C An exemplary modified form of the B1 chimera is shown in SEQ ID NO: 159. The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention comprise a BoNT / AL chain and a H N domain and BoNT / C1 H C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise a BoNT / AL chain and a H domain. N Domains and BoNT / DH C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise a BoNT / AL chain and a H domain. N Domains and BoNT / EH C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise a BoNT / AL chain and a H domain. N Domains and BoNT / FH C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise a BoNT / AL chain and a H domain. N Domains and BoNT / GHC The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise a BoNT / AL chain and a H domain. N Domains and BoNT / XH C The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) may comprise a BoNT / AL chain and a H domain. N Domain and TeNT H C It may include a domain.

[0227] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i. BoNT / BL chains and non-BoNT / BH N and H C domain, ii. BoNT / BH N Domains and non-BoNT / BL chains and H C domain, iii. BoNT / BH C Domains and non-BoNT / BL chains and H N domain, iv. BoNT / BL chain and H N Domains and non-BoNT / BH C domain, v.BoNT / BL chain and H C Domains and non-BoNT / BH N domain, or vi.BoNT / BH N Domain and H C domains and non-BoNT / BL chains.

[0228] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i. BoNT / C1 light chain and non-BoNT / C1 heavy chain N and H C domain, ii. BoNT / C1 H NDomains and non-BoNT / C1 L and H chains C domain, iii. BoNT / C1 H C Domains and non-BoNT / C1 L and H chains N domain, iv. BoNT / C1 L chain and H N Domain and non-BoNT / C1 H C domain, v.BoNT / C1 L chain and H C Domain and non-BoNT / C1 H N domain, or vi.BoNT / C1 H N Domain and H C domain and non-BoNT / C1 light chains.

[0229] Non-limiting examples include BoNT / C1 chimeras in which the non-BoNT / C1 elements are derived from BoNT / D (ie, BoNT / CD chimeras).

[0230] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i. BoNT / DL chain and non-BoNT / DH chain N and H C domain, ii. BoNT / DH N Domains and non-BoNT / DL chains and H C domain, iii. BoNT / DH C Domains and non-BoNT / DL chains and H N domain, iv. BoNT / D L chain and H chain N Domain and non-BoNT / DH C domain, v.BoNT / DL chain and H C Domain and non-BoNT / DH N domain, or vi.BoNT / DH N Domain and HC domain and non-BoNT / DL chain.

[0231] Non-limiting examples include BoNT / D chimeras in which the non-BoNT / D elements are derived from BoNT / C1 (ie, BoNT / DC1 chimeras).

[0232] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i.BoNT / EL chain and non-BoNT / EH N and H C domain, ii.BoNT / EH N Domains and non-BoNT / EL chains and H C domain, iii. BoNT / EH C Domains and non-BoNT / EL chains and H N domain, iv.BoNT / EL chain and H N Domain and non-BoNT / EH C domain, v.BoNT / EL chain and H C Domain and non-BoNT / EH N domain, or vi.BoNT / EH N Domain and H C domains and non-BoNT / EL chains.

[0233] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i. BoNT / FL chain and non-BoNT / FH N and H C domain, ii. BoNT / FH N domains and non-BoNT / FL chains and H C domain, iii. BoNT / FH CDomains and non-BoNT / FL chains and H N domain, iv. BoNT / FL chain and H N Domains and non-BoNT / FH C domain v.BoNT / FL chain and H C Domains and non-BoNT / FH N domain, or vi.BoNT / FH N Domain and H C domains and non-BoNT / FL chains.

[0234] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i. BoNT / GH chain and non-BoNT / GH N and H C domain, ii. BoNT / GH N Domains and non-BoNT / GL chains and H C domain, iii. BoNT / GH C Domains and non-BoNT / GL chains and H N domain, iv. BoNT / GL chain and H N Domains and non-BoNT / GH C domain, v.BoNT / GL chain and H C Domains and non-BoNT / GH N domain, or vi. BoNT / GH N Domain and H C domains and non-BoNT / GL chains.

[0235] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i. BoNT / XL chain and non-BoNT / XH N and HC domain, ii.BoNT / XH N Domains and non-BoNT / XL chains and H C domain, iii.BoNT / XH C Domains and non-BoNT / XL chains and H N domain, iv. BoNT / XL chain and H N Domain and non-BoNT / XH C domain, v.BoNT / XL chain and H C Domain and non-BoNT / XH N domain, or vi.BoNT / XH N Domain and H C domains and non-BoNT / XL chains.

[0236] For example, a clostridial neurotoxin of the present invention (e.g., a modified clostridial neurotoxin containing one or more endosomal protease cleavage sites) can include: i. TeNT L chain and non-TeNT H chain N and H C domain, ii.TeNT H N Domains and non-TeNT L and H chains C domain, iii.TeNT H C Domains and non-TeNT L and H chains N domain, iv. TeNT L chain and H N Domain and non-TeNT H C domain, v. TeNT L chain and H C Domain and non-TeNT H N domain, or vi.TeNT H N Domain and H C domains as well as non-TeNT light chains.

[0237] The term "clostridial neurotoxin" also encompasses newly discovered botulinum neurotoxins and family members of botulinum neurotoxin-like proteins expressed by non-clostridial microorganisms (e.g., the Enterococcus-encoded toxin with closest sequence identity to BoNT / X, the Weissella oryzae-encoded toxin called BoNT / Wo (NCBI Reference Sequence: WP_027699549.1) (cleaving VAMP2 at W89-W90), the Enterococcus faecium-encoded toxin (GenBank: OTO22244.1) (cleaving VAMP2 and SNAP25), the Chryseobacterium pipero-encoded toxin (NCBI Ref. Sequence: WP_034687872.1), and the mosquito BoNT-like protein PMP1 (NCBI Reference Sequence: QEZ70852.1)).

[0238] The term "clostridial neurotoxin" is intended to encompass retargeted clostridial neurotoxins. In the case of retargeted clostridial neurotoxins, the clostridial neurotoxin has been modified to include an exogenous ligand (i.e., not derived from the clostridial neurotoxin) known as a targeting moiety (TM). The TM is selected to confer binding specificity for the desired target cell, and as part of the retargeting process, the native binding moiety of the clostridial neurotoxin (e.g., H C Domain or H CCFor retargeting techniques, see, for example, EP-B-0689459, WO1994 / 021300, EP-B-0939818, US6,461,617, US7,192,596, WO1998 / 007864, EP-B-0826051, US5,989,545, US6,395,513, US6,962,703, WO1996 / 033273, EP-B-0996468, US7,052,702, The clostridial neurotoxins of the present invention are described in WO1999 / 017806, EP-B-1107794, US6,632,440, WO2000 / 010598, WO2001 / 21213, WO2006 / 059093, WO2000 / 62814, WO2000 / 04926, WO1993 / 15766, WO2000 / 61192, and WO1999 / 58571, all of which are incorporated herein by reference in their entirety. Thus, the clostridial neurotoxins of the present invention can be retargeted clostridial neurotoxins. In particular, the modified clostridial neurotoxins of the present invention can be modified retargeted clostridial neurotoxins. The modified retargeted clostridial neurotoxins of the present invention can include a TM displayed at the N- or C-terminus of the single-chain neurotoxin, or the TM can be displayed centrally within the single-chain neurotoxin. In some preferred embodiments, the modified, retargeted Clostridial neurotoxins of the present invention may comprise a TM displayed at the N- or C-terminus of the single-chain neurotoxin.

[0239] Modification of retargeted clostridial neurotoxins may enable the use of TMs that are susceptible to cleavage by proteases (e.g., trypsin, Lys-C, and / or BoNT hydrolase) traditionally used to activate recombinantly produced retargeted clostridial neurotoxins. Thus, modifying a retargeted clostridial neurotoxin to contain one or more endosomal protease activation sites in accordance with the present invention may enable improved stability compared to a corresponding retargeted clostridial neurotoxin activated by a traditional activating protease, such as Lys-C, trypsin, and / or BoNT hydrolase. In some preferred embodiments, the modified retargeted clostridial neurotoxin comprises a BoNT / A light chain (LC / A) and / or a BoNT / A translocation domain (H N / A), particularly preferably LC / A and H N In some preferred embodiments, the modified, retargeted Clostridial neurotoxin comprises both a BoNT / X light chain (LC / X) and / or a BoNT / X translocation domain (H / X). N / X), particularly preferably LC / X and H N / X. Such modified, retargeted BoNT / X are particularly preferred. Non-limiting examples of modified, retargeted Clostridial neurotoxins include those set forth in SEQ ID NOs: 121, 160, 161, and 162.

[0240] The clostridial neurotoxins of the present invention (e.g., modified clostridial neurotoxins) contain a functional H C The polypeptide may lack the native binding function of a clostridial neurotoxin and may lack any functionally equivalent TM. Thus, the polypeptide lacks the native binding function of a clostridial neurotoxin and is unable to bind to rat synaptosomal membranes in a binding assay (Clostridial H C(via a component or any functionally equivalent TM) (as described by Shone et al. (1985) Eur. J. Biochem. 151, 75-82). Preferably, the TM is not a wheat germ agglutinin (WGA) peptide. Thus, in some preferred embodiments, the clostridial neurotoxin is capable of binding to the endogenous H of the clostridial neurotoxin. C or H CC A particularly preferred embodiment is a retargeted Clostridial neurotoxin in which the endogenous H of the Clostridial neurotoxin is replaced by an exogenous TM. C or H CC is a retargeted clostridial neurotoxin in which the TM is replaced by exogenous TM.

[0241] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention are LH N polypeptides (e.g., modified LH N polypeptides), i.e., clostridial L chains and clostridial H chains as defined herein. N It may include a polypeptide comprising or consisting of a domain.

[0242] Clostridial neurotoxins (e.g., modified clostridial neurotoxins) inhibit LH N polypeptides (e.g., modified LH N polypeptide) and a targeting moiety (TM).

[0243] The present invention also encompasses clostridial neurotoxins with additional non-native protease cleavage sites, which require an exogenous protease for cleavage, thereby allowing for improved control over the timing and location of cleavage. Non-native protease cleavage sites that can be used in clostridial neurotoxins include: TEV (Tobacco Etch Virus) (ENLYFQ↓G) (SEQ ID NO: 114) Thrombin (LVPR↓GS) (SEQ ID NO: 115) PreScission (LEVLFQ↓GP) (SEQ ID NO: 116)

[0244] Additional protease cleavage sites include recognition sequences cleaved by non-cytotoxic proteases (e.g., by the light chain of a clostridial neurotoxin). Such sequences include SNARE (e.g., SNAP-25, syntaxin, VAMP) protein recognition sequences cleaved by non-cytotoxic proteases (e.g., the light chain of a clostridial neurotoxin). Clostridial neurotoxins containing non-native protease cleavage sites are described in US 7,132,259, EP 1206554-B2, and US 2007 / 0166332, all of which are incorporated herein by reference in their entireties. The term protease cleavage site also encompasses inteins, which are self-cleaving sequences. Self-splicing reactions can be controlled, for example, by varying the concentration of reducing agents present.

[0245] The present invention also encompasses clostridial neurotoxins that contain a "destructive cleavage site." In such clostridial neurotoxins, a non-native protease cleavage site is incorporated into the clostridial neurotoxin at a location selected such that cleavage at the site reduces or inactivates the activity of the clostridial neurotoxin. The destructive protease cleavage site is susceptible to cleavage by local proteases when the clostridial neurotoxin moves to a non-target location after administration. Suitable non-native protease cleavage sites include those described above. Clostridial neurotoxins that contain destructive cleavage sites are described in WO2010 / 094905 and WO2002 / 044199, both of which are incorporated herein by reference in their entireties.

[0246] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention, particularly the light chain components thereof, may be PEGylated, which can help increase stability (e.g., the duration of action of the light chain component). PEGylation is particularly preferred when the light chain comprises BoNT / A, B, or C1 protease. PEGylation preferably involves adding PEG to the N-terminus of the light chain component. For example, the N-terminus of the light chain may be extended with one or more amino acid (e.g., cysteine) residues, which may be the same or different. One or more of the amino acid residues may have their own PEG molecule attached (e.g., covalently linked). An example of this technology is described in WO2007 / 104567 (incorporated herein by reference in its entirety).

[0247] The chimeric Clostridial neurotoxins of the present invention may not include a therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent) in addition to the light and heavy chains. For example, in one embodiment, the chimeric Clostridial neurotoxin may not include a covalently or non-covalently bound therapeutic or diagnostic agent. Thus, the chimeric Clostridial neurotoxins of the present invention preferably do not function as a delivery vehicle for an additional therapeutic or diagnostic agent.

[0248] In embodiments in which the chimeric Clostridial neurotoxins described herein have a tag (eg, a His tag) and / or a linker for purification, the tag and / or linker are optional.

[0249] The clostridial neurotoxins of the present invention (eg, modified clostridial neurotoxins) may not contain complexing proteins present in the native clostridial neurotoxin complex.

[0250] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention can be produced using recombinant nucleic acid technology. Thus, the modified clostridial neurotoxin (as described above) can be a recombinant modified clostridial neurotoxin. The single-chain clostridial neurotoxin (as described herein) can be a recombinant single-chain neurotoxin.

[0251] The resistance (i.e., reduction of off-target and / or adverse effects) to the modified Clostridial neurotoxins of the present invention may be increased compared to the resistance to the corresponding (unmodified) Clostridial neurotoxin. In particular, the resistance to the modified Clostridial neurotoxins of the present invention may be increased compared to the resistance to the corresponding (unmodified) Clostridial neurotoxin when the unmodified Clostridial neurotoxin is administered (e.g., in a di-chain form). The resistance can be quantified / determined as described below.

[0252] The modified clostridial neurotoxins of the present invention may have equivalent or increased potency compared to the potency of the corresponding (unmodified) clostridial neurotoxin. Specifically, the potency of the modified clostridial neurotoxins of the present invention may be equivalent to or increased compared to the potency of the corresponding (unmodified) clostridial neurotoxin when the unmodified clostridial neurotoxin is administered in a di-chain form. The term "equivalent potency," as used herein, means that the modified clostridial neurotoxin has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, and up to about 100% of the potency of the corresponding (unmodified) clostridial neurotoxin. Preferably, as used herein, "equivalent potency" means that the modified clostridial neurotoxin has at least about 95%, at least about 99%, at least about 100%, at least about 101%, and up to about 105% of the potency of the corresponding (unmodified) clostridial neurotoxin. As used herein, the term "increased potency" means that the modified clostridial neurotoxin has at least about 10%, at least about 15%, at least about 20%, or at least about 25% greater potency than the potency of the corresponding (unmodified) clostridial neurotoxin. Potency can be measured using any suitable assay, conventional examples of which are described herein.

[0253] The modified clostridial neurotoxins of the present invention typically have an improved safety profile and / or therapeutic window compared to the safety profile and / or therapeutic window of the corresponding (unmodified) clostridial neurotoxin. Without being bound by theory, this may be due to improved tolerance and / or equivalent or increased efficacy. In particular, the modified clostridial neurotoxins of the present invention have an improved safety profile and / or therapeutic window compared to the safety profile and / or therapeutic window of the corresponding (unmodified) clostridial neurotoxin when the unmodified clostridial neurotoxin is administered (e.g., in a di-chain form).

[0254] One way to define these advantageous properties (corresponding to an increased therapeutic index) can be in terms of the safety factor (for clinical use) or tolerability index (TI, in the case of animal models (which can be calculated as described below)) of the modified clostridial toxin. In this regard, undesirable effects of a clostridial neurotoxin (e.g., effects caused by diffusion of the neurotoxin away from the site of administration) can be experimentally evaluated by measuring the percentage of weight loss in a relevant animal model (e.g., mice, where weight loss is detected within 7 days of administration). The desired on-target effect of a clostridial toxin can be experimentally evaluated by any suitable technique, depending on the target cell of interest. Suitable assays are known in the art, and it would be routine for one of skill in the art to select an appropriate assay for a given target cell type. For clostridial neurotoxins of the present invention that target motor neurons, the Digital Abduction Score (DAS) assay, a measure of muscle paralysis, can be used. The DAS assay can be performed by injecting 20 μl of a (modified) clostridial toxin formulated in gelatin phosphate buffer into the gastrocnemius / soleus muscle complex of a mouse, followed by assessing the digit abduction score using the method of Aoki (Aoki KR, Toxicon 39:1815-1820; 2001). In the DAS assay, the mouse's tail is briefly suspended to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. After injection of the clostridial toxin, the degree of digit abduction is scored on a 5-point scale (0 = normal to 4 = maximal reduction in digit abduction and leg extension). For clostridial neurotoxins of the present invention that target other neuronal subtypes, any suitable assay method known in the art can be used. SNARE cleavage assays can also be used to assess the activity of the modified clostridial neurotoxins of the present invention, examples of which are well described in the art (e.g., Western blot). Assays to detect and / or quantify the effect of modified Clostridial neurotoxins on the release of marker signaling molecules can also be used.The specific marker signaling molecule can be selected depending on the cell type(s) targeted by the modified Clostridial neurotoxin. For example, the signaling molecule can be a hormone, substance P, CGRP, glutamate, or glycine, depending on whether cells involved in hormone secretion or pain-sensing neurons are targeted. For pain treatment, animal tests can be used to assess whether a higher tolerance to noxious stimuli is observed. Typical in vivo assays measure different types of pain (e.g., mechanical, cold, heat), and the readout can be behavioral (e.g., licking / biting the treated area or withdrawal from the noxious stimulus) or involve the use of the Von Frey test. Any suitable nociceptive test can be used, and examples of such tests are well known in the art.

[0255] The safety factor or TI of a clostridial neurotoxin can then be expressed as the ratio of the amount of toxin required to produce a 10% loss in body weight (peak effect measured within 7 days of administration in mice) to the amount of toxin required to produce a DAS score of 2. A high safety factor or TI score is therefore desirable and indicates a toxin that can effectively paralyze target muscles with few undesired off-target effects. The modified toxins of the present invention may have a safety factor and / or TI that is higher than that of a comparable unmodified (pre-modification) single-chain clostridial neurotoxin. Calculation of TI may vary depending on the experimental model used.

[0256] For example, in a DAS mouse model, a modified Clostridial toxin of the invention has a TI of at least 8 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), where TI is calculated as follows: - dose of toxin required for a 10% change in body weight (pg / mouse) ÷ DAS ED 50 (pg / mouse) [ED 50 = dose required to obtain a DAS score of 2].

[0257] For clinical use, a safety margin can be calculated.

[0258] The present invention provides nucleic acids (e.g., DNA or RNA) comprising a nucleic acid sequence encoding a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) described herein. The nucleic acid sequence can be prepared as part of an expression vector in which the nucleic acid is operably linked to a promoter. Preferably, the nucleic acid can be prepared as part of a DNA expression vector comprising a promoter and a terminator.

[0259] Preferably, the vector has a promoter selected from the following: Promoter Inducer Typical induction conditions Tac (hybrid) IPTG 0.2mM (0.05-2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2mM (0.05-2.0mM)

[0260] Alternatively, the promoter may preferably be selected from: Promoter Inducer Typical induction conditions Tac (hybrid) IPTG 0.2mM (0.05-2.0mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2mM (0.05-2.0mM) T5-lac operator IPTG 0.2mM (0.05-2.0mM)

[0261] The nucleic acid molecule of the present invention can be produced by any suitable process known in the art.Therefore, the nucleic acid molecule can be produced by chemical synthesis techniques.Alternatively, the nucleic acid molecule of the present invention can be produced by molecular biology techniques.

[0262] The nucleic acid molecules and expression vectors of the present invention are preferably designed in silico and then synthesized by conventional synthetic techniques (including conventional DNA synthesis techniques).

[0263] The above nucleic acid sequence information is optionally modified for codon bias according to the expression system of the final host cell (eg, E. coli) used.

[0264] The present invention provides nucleotide sequences encoding the modified Clostridial neurotoxins of the present invention. The nucleotide sequences of the present invention encode polypeptides that include one or more endosomal protease cleavage sites as described herein.

[0265] The nucleotide sequence can comprise a sequence having at least 70% sequence identity to SEQ ID NO: 163, wherein the nucleic acid encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced with a nucleic acid encoding an extrinsic activation loop comprising one or more endosomal protease sites or the one or more endosomal cleavage sites. The nucleotide sequence can comprise a sequence having at least 80% or 90% sequence identity to SEQ ID NO: 163, wherein the nucleic acid encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced with a nucleic acid encoding an extrinsic activation loop comprising one or more endosomal protease sites or the one or more endosomal cleavage sites. Preferably, the nucleotide sequence comprises (more preferably consists of) SEQ ID NO: 163, wherein the nucleic acid encoding the BoNT / C activation loop (SEQ ID NO: 164) is replaced with a nucleic acid encoding an extrinsic activation loop comprising one or more endosomal protease sites or the one or more endosomal cleavage sites. Non-limiting examples of nucleic acids encoding extrinsic activation loops that can replace SEQ ID NO: 164 in SEQ ID NO: 163 include SEQ ID NOs: 165, 166, and 167. Thus, non-limiting examples of nucleic acids encoding exemplary modified Clostridial neurotoxins (particularly modified retargeted BoNT / Xs) include SEQ ID NOs: 168, 169, and 170.

[0266] The nucleotide sequence may encode a modified Clostridial neurotoxin having at least 70% sequence identity to one or more of SEQ ID NOs: 121 or 159-162. The nucleotide sequence may encode a modified Clostridial neurotoxin having at least 80% or 90% sequence identity to one or more of SEQ ID NOs: 121 or 159-162. Preferably, the nucleotide sequence encodes a modified Clostridial neurotoxin comprising (more preferably consisting of) any one of SEQ ID NOs: 121 or 159-162.

[0267] The terms "nucleotide sequence" and "nucleic acid" and "polynucleotide" are used interchangeably herein. Preferably, the nucleotide sequence is a DNA sequence.

[0268] The present invention provides a method for producing a single-chain (modified) Clostridial neurotoxin protein having a light chain and a heavy chain, the method comprising expressing a polynucleotide or expression vector described herein in a suitable host cell and recovering the expressed modified Clostridial neurotoxin. Recovering the expressed modified Clostridial neurotoxin may comprise lysing the host cell to obtain a host cell homogenate containing the single-chain (modified) Clostridial neurotoxin protein and / or isolating the single-chain (modified) Clostridial neurotoxin protein. The method may further comprise the step of introducing the polynucleotide or expression vector described herein into the host cell. Suitable host cells include bacterial cell lines used for recombinant production of Clostridial neurotoxins, particularly Escherichia coli cells.

[0269] The present invention provides a method for proteolytically cleaving the (modified) clostridial neurotoxin of the present invention into the corresponding di-chain clostridial neurotoxin, which method comprises contacting the (modified) clostridial neurotoxin with one or more endosomal proteases to produce a di-chain clostridial neurotoxin (e.g., one in which the light chain and heavy chain are linked together by a disulfide bond).

[0270] Therefore, the present invention provides a di-chain clostridial neurotoxin obtainable by the method of the present invention.

[0271] As used herein, the term "obtainable" also encompasses the term "obtained." Preferably, the term "obtainable" means obtained.

[0272] Activation of modified clostridial neurotoxins The present invention provides a method for proteolytically cleaving the modified clostridial neurotoxin of the present invention into the corresponding di-chain clostridial neurotoxin, which method comprises contacting the modified clostridial neurotoxin with one or more endosomal proteases to produce the di-chain clostridial neurotoxin. The contacting can be in vitro, ex vivo, or in vivo, preferably in vivo. Thus, the therapeutic methods and uses of the present invention can include in vivo activation of the modified clostridial neurotoxin of the present invention by cleaving it at one or more endosomal protease activation sites by expressing one or more endosomal proteases in target cells.

[0273] Thus, the methods of the present invention can further comprise contacting the modified Clostridial neurotoxin with one or more endosomal proteases to produce the corresponding double-chain modified Clostridial neurotoxin. Preferably, the contacting occurs in vivo.

[0274] The present invention also provides a method for proteolytically cleaving a single-chain Clostridial neurotoxin into a corresponding di-chain Clostridial neurotoxin, the method comprising: (a) providing a single-chain Clostridial neurotoxin; and (b) contacting the single-chain Clostridial neurotoxin with one or more endosomal proteases, wherein the single-chain Clostridial neurotoxin has an activation loop that includes or consists of one or more endosomal protease cleavage sites described herein (e.g., SEQ ID NOs: 1-38, 130-152, or 171-187; 12-38, 130-152, or 171-187; 1-34, 130-152, or 171-187; or any one or more of 12-34, 130-152, or 171-187), and the one or more endosomal proteases hydrolyze a peptide bond in the activation loop to produce a di-chain Clostridial neurotoxin. Preferably, the contacting occurs in vivo.

[0275] The present invention encompasses contacting a single-chain clostridial neurotoxin (e.g., a modified clostridial neurotoxin of the present invention) with one or more endosomal proteases, where the one or more endosomal proteases are capable of hydrolyzing a peptide bond within the activation loop of the single-chain clostridial neurotoxin to produce a di-chain clostridial neurotoxin. Preferably, the contacting occurs in vivo.

[0276] The contacting can occur under any suitable conditions such that more than 30%, 40%, 50%, or 60% (preferably more than 70%) of the single-chain clostridial neurotoxins are proteolytically processed to the corresponding di-chain clostridial neurotoxins without or substantially without hydrolysis of peptide bonds outside the activation loop of the clostridial neurotoxins. "Without substantial hydrolysis" can mean that less than 5%, 4%, 3%, 2%, or 1% of the contacted clostridial neurotoxins contain peptide bonds outside the activation loop that are hydrolyzed by one or more endosomal proteases in the methods of the invention.

[0277] Those skilled in the art can select appropriate reaction times, temperatures, buffers, and molar ratios of protease to single-chain Clostridial neurotoxin to achieve the above. Optimization of such conditions can be determined empirically using routine techniques, such as visual analysis of the reaction products after contact by SDS-PAGE (e.g., staining with Coomassie or similarly sensitive dyes) or spectroscopic techniques (e.g., mass spectrometry).

[0278] The methods of the invention preferably result in the production of only clostridial neurotoxin light and heavy chains, as assessed by SDS-PAGE (eg, stained with Coomassie or similarly sensitive dyes).

[0279] Proteolytic processing by one or more endosomal proteases in the methods of the invention typically results in the production of fewer than five degradation products of the clostridial neurotoxin light or heavy chain, more preferably fewer than four, three, two, or one degradation product. Preferably, the light and heavy chains produced by the methods of the invention are full-length light and heavy chains.

[0280] Typically, treatment with each of the one or more endosomal proteases in the method of the present invention hydrolyzes 5 or less, 4 or less, 3 or less, 2 or less, or 1 peptide bond in the modified clostridial neurotoxin, preferably 1 or 2 peptide bonds. Preferably, treatment with each of the one or more endosomal proteases in the method of the present invention hydrolyzes 5 or less, 4 or less, 3 or less, 2 or less, or 1 peptide bond in the activation loop of the modified clostridial neurotoxin, preferably 1 or 2 peptide bonds. When two or more endosomal protease cleavage sites are present in the modified clostridial neurotoxin of the present invention, the total number of peptide bonds that can be hydrolyzed by the two or more endosomal proteases is typically 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Preferably, one peptide bond is hydrolyzed by each of the two or more endosomal proteases. Exemplary endosomal protease cleavage sites are described herein along with the location of the peptide bond that is hydrolyzed.

[0281] For in vitro activation of modified clostridial neurotoxins with one or more endosomal proteases of the present invention, any suitable conditions for activation can be used. Determining suitable conditions is within the routine skill of one of ordinary skill in the art. As a non-limiting example, about 2 μg to about 5 μg of cathepsin L can be used per 0.1 to 1 mg of modified clostridial neurotoxin, with activation occurring at room temperature (about 21°C) for 1 to 3 hours. As a further non-limiting example, about 10 μg to about 25 μg of AEP can be used per 0.1 to 1 mg of modified clostridial neurotoxin, with activation occurring at room temperature (about 21°C) for 1 to 3 hours. Other temperatures (e.g., about 4°C or about 37°C) may also be used, with the amount of endosomal protease being increased or decreased accordingly.

[0282] Many cells endogenously express one or more endosomal proteases in endosomes and / or lysosomes. As used herein, the term endosome encompasses lysosomes. Thus, one or more endosomal proteases expressed by a cell are typically present within the cell. Thus, the step of contacting a clostridial neurotoxin with one or more endosomal proteases according to the present invention can occur within a cell treated with the clostridial neurotoxin. In other words, contacting a clostridial neurotoxin with one or more endosomal proteases according to the present invention can involve one or more endosomal proteases endogenously present in the target cell. Thus, contacting a clostridial neurotoxin with one or more endosomal proteases according to the present invention can occur in vivo after administration of a clostridial neurotoxin to an individual. When the contacting step occurs in vivo, it typically involves one or more endosomal proteases endogenously present in one or more cells present in the tissue or organ treated according to the present invention.

[0283] The present invention also provides di-chain clostridial neurotoxins obtainable by the methods of the present invention. Because activation to the di-chain form occurs by cleavage at one or more endosomal protease cleavage sites as described herein, the cleaved C- and N-terminal ends of the resulting di-chain clostridial neurotoxin differ in sequence compared to the corresponding (unmodified) clostridial neurotoxin. In contrast, conventional trypsin cleavage of (unmodified) BoNT / A yields a di-chain having an LC with a C-terminus ending in the sequence TSK and an HC with an N-terminus beginning with ALNDLC. These di-chain clostridial neurotoxins can be used for the treatments described herein. All disclosures herein regarding therapeutic indications and formulations in the context of the modified or single-chain clostridial neurotoxins of the present invention apply equally and without reservation to the di-chain clostridial neurotoxins obtainable by the methods of the present invention, unless otherwise specified.

[0284] Treatments and Formulations The clostridial neurotoxins of the present invention are preferably useful in pharmaceutical and / or cosmetic applications. In use, the modified clostridial neurotoxins of the present invention can be cleaved in vivo by one or more endosomal proteases described herein, so the clostridial neurotoxins are preferably administered in a single-chain form. Alternatively, the modified clostridial neurotoxins of the present invention (e.g., obtained by the method of the present invention) may be administered in a two-chain form.

[0285] The (modified) Clostridial neurotoxins of the present invention can be used for the prevention or treatment of certain medical or cosmetic diseases and conditions. Thus, in a further aspect, the present invention provides the above-mentioned (modified) Clostridial neurotoxins for use in medicine. Furthermore, as described herein, the present invention relates to single-chain Clostridial neurotoxins for use in the prevention or treatment of certain medical or cosmetic diseases and conditions, wherein the single-chain Clostridial neurotoxins are administered to a subject. Furthermore, as described herein, the present invention relates to double-chain Clostridial neurotoxins obtainable by the methods of the present invention for use in the prevention or treatment of certain medical or cosmetic diseases and conditions, wherein the double-chain Clostridial neurotoxins obtainable by the methods of the present invention are administered to a subject. Thus, in a further aspect, the present invention provides the above-mentioned (modified) Clostridial neurotoxins for use in medicine.

[0286] Thus, the present invention is directed to the treatment of conditions associated with unwanted immune secretions, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapeutic (cosmetic) applications that benefit from cell / muscle inactivation (via SNARE downregulation or inactivation), neuromuscular disorders or conditions of eye movement (e.g., conjugate strabismus, vertical strabismus, lateral rectus palsy, nystagmus, myopathy due to thyroid abnormalities), writer's cramp, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasticity, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasticity, muscle spasms or stiffness (charley horse), levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), forehead grooves, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders. In some cases, the condition may be selected from phantom pain (e.g., phantom limb pain) and bladder pain syndrome. Similarly, the present invention also relates to single-chain and double-chain clostridial neurotoxins obtainable by the methods of the present invention, for use in the treatment or prevention of the above-mentioned diseases or conditions. Preferably, the compositions of the present invention may be used for the prevention or treatment of a disease or condition selected from limb spasticity (upper or lower limb), cervical dystonia, headache disorders (preferably migraine), blepharospasm, hemifacial spasm, and lower urinary tract disorders (e.g., bladder pain syndrome (preferably interstitial cystitis)), overactive bladder, and detrusor overactivity (e.g., neurogenic detrusor overactivity).

[0287] When a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) of the present invention comprises a BoNT / X sequence (or a portion thereof), the clostridial neurotoxin may be able to target other types of secretory cells besides neurons due to its ability to cleave VAMP4, VAMP5, and / or Ykt6. In some embodiments, the targeted secretory cells are secretory immune cells. As used herein, "secretory immune cells" refer to immune cells that secrete cytokines, chemokines, or antibodies. Such secretory immune cells may be innate immune cells, including, but not limited to, natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. Antibody-secreting secretory immune cells (e.g., leukocytes) may also be targeted by the clostridial neurotoxins of the present disclosure. Non-limiting examples of antibody-secreting cells include, but are not limited to, plasma B cells, plasmocytes, plasmacytes, and effector B cells. In some embodiments, the clostridial neurotoxin can modulate the immune response. Thus, the present invention further contemplates the therapeutic use of the clostridial neurotoxin of the present invention for treating conditions associated with unwanted secretions, preferably unwanted immune secretions. Conditions associated with unwanted immune secretions include, but are not limited to, inflammation, psoriasis, allergies, hemophagocytic lymphohistiocytosis, and alcoholic pancreatic disease.

[0288] The present invention also provides the use of a Clostridial neurotoxin as described above (e.g., a modified Clostridial neurotoxin or a two-chain Clostridial neurotoxin obtainable by the methods of the present invention) in the manufacture of a medicament for use in a method for preventing or treating a disease or disorder described herein.

[0289] The present invention also provides a method for treating a disease or disorder described herein, the method comprising administering a therapeutically effective amount of a clostridial neurotoxin as described above (e.g., a modified clostridial neurotoxin or a two-chain clostridial neurotoxin obtainable by the method of the present invention) to a subject in need thereof.

[0290] The present invention also provides non-therapeutic uses of the compositions described herein for treating aesthetic or cosmetic conditions. For cosmetic or aesthetic uses, it is preferred that the individual being treated does not suffer from any of the aforementioned diseases or disorders associated with undesired neuronal activity. More preferably, the individual is healthy, i.e., does not suffer from any disease. Preferably, the compositions of the present invention can be used to prevent or treat upper facial wrinkles (glabellar lines, crow's feet, and / or intrathecal lines).

[0291] The present invention provides pharmaceutical compositions comprising a (modified) clostridial neurotoxin or a two-chain clostridial neurotoxin of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt. Preferably, the (modified) clostridial neurotoxin is in a single-chain form (e.g., modified to contain one or more endosomal protease cleavage sites). The pharmaceutical compositions of the present invention may be liquid compositions (or formulations) or solid compositions (or formulations).

[0292] The present invention also provides a cosmetic composition comprising the (modified) Clostridial neurotoxin of the present invention or the two-chain Clostridial neurotoxin of the present invention and a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant, and / or salt. The present invention also provides the use of a cosmetic composition comprising a Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin or two-chain Clostridial neurotoxin obtainable by the method of the present invention) to prevent or alleviate a cosmetic indication indicating the need for the application of a botulinum neurotoxin. The present invention also provides the use of a cosmetic composition comprising a Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin or two-chain Clostridial neurotoxin obtainable by the method of the present invention) to prevent or alleviate a cosmetic indication indicating the need for the application of a botulinum neurotoxin. Preferably, the (modified) Clostridial neurotoxin is in a single-chain form (e.g., modified to contain one or more endosomal protease cleavage sites). The cosmetic composition of the present invention may be a liquid composition (or formulation) or a solid composition (or formulation).

[0293] The clostridial neurotoxins (e.g., modified clostridial neurotoxins) of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation, or topical application. Compositions suitable for injection may take the form of a solution, suspension, or emulsion, or may be a dry powder that is dissolved or suspended in a suitable vehicle before use.

[0294] The liquid compositions of the present invention can be (i) pre-lyophilized solutions, (ii) reconstituted solutions, or (iii) solutions not intended for lyophilization and / or not reconstituted after lyophilization. Liquid compositions in category (iii) are also referred to as "ready-to-use" compositions or "ready-to-use" solutions because they are manufactured and formulated as liquids and sold for use in liquid form. All disclosures herein relating to liquid formulations apply to any liquid formulation (including pre-lyophilized solutions, reconstituted solutions, and ready-to-use compositions) unless expressly stated to the contrary. Liquid compositions can be packaged based on the amount (e.g., absolute weight) of the chimeric Clostridial neurotoxin of the present invention described herein. Liquid compositions can be packaged to allow for up to 15 injections from a single container.

[0295] The solid composition may be packaged based on the amount (particularly by absolute weight) of the modified Clostridial neurotoxin of the present invention described herein.

[0296] In the case of a locally delivered Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin), the Clostridial neurotoxin (e.g., a modified Clostridial neurotoxin) can be formulated as a cream (e.g., for topical application) or for subcutaneous injection.

[0297] Local delivery means can include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of clostridial neurotoxins (e.g., modified clostridial neurotoxins) allow for delivery to the lungs and / or other nasal passages and / or bronchi or airway passages.

[0298] The clostridial neurotoxins (eg, modified clostridial neurotoxins) of the present invention can be administered to a patient by intrathecal or epidural injection in the spinal cord at the level of the spinal segment involved in innervating the affected organ.

[0299] Preferred routes of administration are via laparoscopy and / or local (especially intramuscular) injection.

[0300] The dosage ranges for administering the compositions of the present invention are those to achieve the desired therapeutic effect. A therapeutically effective dose refers to the amount of chimeric neurotoxin used in the compositions of the present invention that prevents, alleviates, or treats symptoms associated with the diseases or conditions referred to herein. The therapeutic efficacy and toxicity of compounds are typically determined in the art using standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 The dose ratio between therapeutic and toxic effects is the therapeutic index, and the LD 50 / ED 50 It will generally be understood that the dosage range required for a given pharmaceutical agent will depend on the precise nature of the composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent, or severity of the patient's condition, any contraindications, and the judgment of the attending physician.

[0301] In the context of the modified neurotoxin compositions of the present invention, suitable single unit doses (also referred to as unit doses), i.e., the dose administered per injection site, are described in the art, for example, in WO2021 / 186160, WO2021 / 186167, WO2023 / 047127, WO2023 / 089343, and WO2023 / 041934, each of which is incorporated herein by reference in its entirety. By way of non-limiting example, one unit dose is 15,000 pg of modified neurotoxin, 25,000 pg of modified neurotoxin, or 36,000 pg of modified neurotoxin.

[0302] Treatment may involve injection at multiple injection sites (typically no more than 20, preferably no more than 15 injection sites), with each injection site receiving one unit dose.

[0303] Fluid dosage forms are typically prepared using a clostridial neurotoxin (e.g., a modified clostridial neurotoxin) and a pyrogen-free sterile vehicle. The clostridial neurotoxin (e.g., a modified clostridial neurotoxin) can be dissolved or suspended in the vehicle depending on the vehicle and concentration used. When preparing a solution, the clostridial neurotoxin (e.g., a modified clostridial neurotoxin) is dissolved in the vehicle, the solution is made isotonic by adding sodium chloride if necessary, and sterilized by filtration through a sterile filter using aseptic techniques, and then filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the solution is sufficiently stable, the solution in a sealed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives, or bactericides, suspending agents, or emulsifiers, and / or local anesthetics can be dissolved in the vehicle.

[0304] Dry powders (dissolved or suspended in a suitable vehicle prior to use) can be prepared by filling pre-sterilized ingredients into sterile containers using aseptic techniques in a sterile area. Alternatively, the ingredients may be dissolved in suitable containers using aseptic techniques in a sterile area. The product is then lyophilized and the container is aseptically sealed.

[0305] Parenteral suspensions suitable for intramuscular, subcutaneous, or intradermal injection are prepared in substantially the same manner, except that the sterile components are suspended in a sterile vehicle rather than dissolved, and sterilization by filtration cannot be accomplished. The components can be isolated under sterile conditions, or alternatively, can be sterilized after isolation (e.g., by gamma irradiation).

[0306] Advantageously, a suspending agent (eg, polyvinylpyrrolidone) is included in the composition(s) to promote uniform distribution of the components.

[0307] Administration according to the present invention can utilize a variety of delivery technologies, including microparticle encapsulation, viral delivery systems, or high-pressure aerosol impaction.

[0308] It is intended that disclosures relating to the various methods of the present invention apply equally to other methods, clostridial neurotoxins, e.g., modified clostridial neurotoxins (whether single-chain or double-chain), uses, or pharmaceutical compositions, and medical uses thereof, and vice versa.

[0309] sequence homology Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global, local, and hybrid (e.g., segment approach) methods. Protocols for determining percent identity are routine procedures within the skill of those in the art. Global methods align the sequences from beginning to end of the molecule and determine the best alignment by summing the scores of individual residue pairs and applying gap penalties. Non-limiting examples of methods include 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. MoI. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-limiting methods include, for example, 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., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214(1993)), and 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)).

[0310] Thus, percent sequence identity is determined by conventional methods. See, e.g., 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 and the alignment score is optimized 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 standard one-letter codes):

[0311] 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, percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. Sequence comparison and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are well known to those skilled in the art.

[0312] Alignment scores for determining sequence identity [ka]

[0313] The percent identity is then calculated as follows: Total number of identical matches ___________________________________×100 [Length of the longer sequence + [Number of gaps introduced into the longer sequence to align the two sequences]

[0314] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, small deletions (typically deletions of 1 to about 30 amino acids), and small amino- or carboxyl-terminal extensions (e.g., an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag).

[0315] Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid Aspartic acid Polarity: Glutamine Asparagine Hydrophobic: Leucine Isoleucine Balin Aromatic: Phenylalanine Tryptophan Tyrosine Low molecular weight: glycine Alanine Serine Threonine methionine

[0316] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues in the polypeptides. The polypeptides of the invention may also include unnatural amino acid residues.

[0317] Unnatural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating unnatural amino acid residues into proteins are known in the art. For example, nonsense mutations can be suppressed using chemically aminoacylated suppressor tRNAs in an in vitro system. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing E. coli S30 extracts and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, e.g., 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 mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996). Within the third method, E. coli cells are cultured in the absence of the replacing natural amino acid (e.g., phenylalanine) and in the presence of the desired unnatural amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnatural amino acid is incorporated into the polypeptide in place of its natural counterpart.See Koide et al., Biochem. 33:7470-6, 1994. Natural amino acid residues can be converted to non-natural 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).

[0318] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, unnatural amino acids, and artificial amino acids can be substituted for amino acid residues in the polypeptides of the present invention.

[0319] Essential amino acids in the polypeptides of the 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 structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, coupled with mutations of putative contact site amino acids. See, e.g., 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. Essential amino acid content can also be inferred from analysis of homology with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0320] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, 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 within a polypeptide, selecting functional polypeptides, and sequencing the mutagenized polypeptides to determine the spectrum of permissible 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 No. WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0321] Sequence information Where the first Met amino acid residue or the corresponding first codon is shown in any of the following SEQ ID NOs, that residue / codon is optional. SEQ ID NO: 1: Cathepsin L cleavage site consensus sequence Leu Xaa1Xaa2Xaa3Xaa4Xaa5Xaa6 wherein Xaa1 is L, V, F, I, or Y; Xaa2 is G, K, or A (preferably G or K); Xaa3 is A, G, or S; Xaa4 is P or E, Xaa5 is P or G, Xaa6 is P, D, or E. SEQ ID NO: 2: Cathepsin L cleavage site consensus sequence Xaa1Xaa2Xaa3Xaa4Xaa5Xaa6 In the sequence, Xaa1 is G or M, Xaa2 is F, V, Y, I, or L; Xaa3 is G, Q, or T (preferably G or Q); Xaa4 is G or H (G is preferred); Xaa5 is P or H, Xaa6 is H, P, or G. SEQ ID NO: 3: Cathepsin L cleavage site consensus sequence Leu Xaa1Xaa2Xaa3Xaa4Xaa5Xaa6 wherein Xaa1 is L, V, F, I, or Y; Xaa2 is G or K, Xaa3 is A, G, or S; Xaa4 is P or E, Xaa5 is P or G, Xaa6 is P, D, or E. SEQ ID NO: 4: Cathepsin L cleavage site consensus sequence Xaa1Xaa2Xaa3Gly Xaa4Xaa5 In the sequence, Xaa1 is G or M, Xaa2 is F, V, Y, I, or L; Xaa3 is G or Q, Xaa4 is P or H, Xaa5 is H, P, or G. SEQ ID NO: 5: Cathepsin B cleavage site consensus sequence Leu Xaa1Xaa2Xaa3Xaa4Xaa5Gly In the sequence, Xaa1 is A, V, or F; Xaa2 is G or A (preferably G); Xaa3 is G, L, or F; Xaa4 is A or V; Xaa5 is G or A (G is preferred). SEQ ID NO: 6: Cathepsin B cleavage site consensus sequence Xaa1Xaa2Gly Xaa3Xaa4Gly In the sequence, Xaa1 is G, L, or P; Xaa2 is A, V, F, or Y; Xaa3 is F or G (F is preferred); Xaa4 is V or A. SEQ ID NO: 7: Cathepsin B cleavage site consensus sequence Leu Xaa1Gly Xaa2Xaa3Gly Gly In the sequence, Xaa1 is A, V, or F; Xaa2 is G, L, or F; Xaa3 is A or V. SEQ ID NO: 8: Cathepsin B cleavage site consensus sequence Xaa1Xaa2Gly Phe Xaa3Gly In the sequence, Xaa1 is G, L, or P; Xaa2 is A, V, F, or Y; Xaa3 is V or A. SEQ ID NO: 9: Cathepsin D cleavage site consensus sequence Leu Xaa1Xaa2Xaa3Xaa4Xaa5Xaa6 In the sequence, Xaa1 is E or L, Xaa2 is V or E (V is preferred); Xaa3 is L or F, Xaa4 is I, L, or F; Xaa5 is V or A, Xaa6 is L or E. SEQ ID NO: 10: Cathepsin D cleavage site consensus sequence Leu Xaa1Val Xaa2Xaa3Xaa4Xaa5 In the sequence, Xaa1 is E or L, Xaa2 is L or F, Xaa3 is I, L, or F; Xaa4 is V or A; Xaa5 is L or E. SEQ ID NO: 11: AEP cleavage site consensus sequence Xaa1Xaa2Glu Xaa3Xaa4Glu Xaa5 In the sequence, Xaa1 is E or A, Xaa2 is A or G, Xaa3 is N or D, Xaa4 is G or S; Xaa5 is L or A. SEQ ID NO: 12: Cathepsin L cleavage site STSQKSIVAYTMSLGADSS SEQ ID NO: 13: Cathepsin L cleavage site LFRGGHHPD SEQ ID NO: 14: Cathepsin L cleavage site ELVTPARDFGHFGLS SEQ ID NO: 15: Cathepsin L cleavage site QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQA SEQ ID NO: 16: Cathepsin L cleavage site STSQKSIVAYTMSLGADSSTGFGTNE SEQ ID NO: 17: Cathepsin L cleavage site LFRGGHHPDTGFGTNE SEQ ID NO: 18: Cathepsin L cleavage site ELVTPARDFGHFGLSTGFGTNE SEQ ID NO: 19: Cathepsin L cleavage site QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNE SEQ ID NO: 20: Cathepsin B cleavage site LFGFVG SEQ ID NO: 21: Cathepsin D cleavage site ALVEKLLELKKK SEQ ID NO: 22: AEP cleavage site QEAANERQQ SEQ ID NO: 23: AEP cleavage site SGLTNIKTE SEQ ID NO: 24: AEP cleavage site PDLKNVKSK SEQ ID NO: 25: AEP cleavage site PGGGNKKIE SEQ ID NO: 26: AEP cleavage site QLGKNEEGA SEQ ID NO: 27: Cathepsin L cleavage site QKVGKAMYAP SEQ ID NO: 28: Cathepsin B cleavage site GFLG SEQ ID NO: 29: Cathepsin D cleavage site TVIVITLVMLKKKQ SEQ ID NO: 30: Cathepsin D cleavage site PVETDSEEQPYLEMDL SEQ ID NO: 31: Cathepsin D cleavage site LEGMELIVSQVHPETKENEIYPVWSGLP SEQ ID NO: 32: Cathepsin D cleavage site QKEYALLYKLDIEP SEQ ID NO: 33: Cathepsin D cleavage site SLAEEEVVIRSED SEQ ID NO: 34: AEP cleavage site ERNSNLVGAA SEQ ID NO: 35: Multiple endosomal protease cleavage sites [ka] The bold and underlined residues indicate the P1 residues within the endosomal protease cleavage site. SEQ ID NO: 36: Multiple endosomal protease cleavage sites [ka] The bold and underlined residues indicate the P1 residues within the endosomal protease cleavage site. SEQ ID NO: 37: Multiple endosomal protease cleavage sites [ka] The bold and underlined residues indicate the P1 residues within the endosomal protease cleavage site. SEQ ID NO: 38: Multiple endosomal protease cleavage sites [ka] SEQ ID NO: 39: Human cathepsin L1 amino acid sequence (UniProt accession number P07711) MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNEEGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKGKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSEQNL VDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV SEQ ID NO: 40: Human cathepsin B amino acid sequence (UniProt accession number P07858) MWQLWASLCCLLVLANARSRPSFHPLSDELVNYVNKRNTTWQAGHNFYNVDMSYLKRLCGTFLGGPKPPQRVMFTEDLKLPASFDAREQWPQCPTIKEIRDQGSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLTCCGSMCGDGCNGGYPAEAWNFWTRKGLVS GGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPTYKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMGGHAIRILGWGVENGTPYWLVANSWNTDWGDNGFFKILRGQDHCGIESEVVAGIPRTDQYWEKI SEQ ID NO: 41: Human cathepsin D amino acid sequence (UniProt accession number P07339) MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVPAVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNL WVPSIHCKLLDIACWIHHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFGEATKQPGITFIAAKFDGILGMAYPRI SVNNVLPVFDNLMQQKLVDQNIFSFYLSRDPDAQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAIVDTGTSLMVGPVDEVR ELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQAGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARL SEQ ID NO: 42: Human cathepsin K amino acid sequence (UniProt accession number P43235) MWGLKVLLLPVVSFALYPEEILDTHWELWKKTHRKQYNNKVDEISRRLIWEKNLKYISIHNLEASLGVHTYELAMNHLGDMTSEEVVQKMTGLKVPLSHSRSNDTLYIPEWEGRAPDSVDYRKKGYVTPVKNQGQCGSCWAFSSVGALEGQLKKKTGKLLNLSP QNLVDCVSENDGCGGGYMTNAFQYVQKNRGIDSEDAYPYVGQEESCMYNPTGKAAKCRGYREIPEGNEKALKRAVARVGPVSVAIDASLTSFQFYSKGVYYDESCNSDNLNHAVLAVGYGIQKGNKHWIIKNSWGENWGNKGYILMARNKNNACGIANLASFPKM SEQ ID NO: 43: Human cathepsin S amino acid sequence (UniProt accession number P25774) MKRLVCVLLVCSSAVAQLHKDPTLDHHWHLWKKTYGKQYKEKNEEAVRRLIWEKNLKFVMLHNLEHSMGMHSYDLGMNHLGDMTSEEVMSLMSSLRVPSQWQRNITYKSNPNRILPDSVDWREKGCVTEVKYQGSCGACWAFSAVGALEAQLKLKTGKLVSLSAQ NLVDCSTEKYGNKGCNGGFMTTAFQYIIDNKGIDSDASYPYKAMDQKCQYDSKYRAATCSKYTELPYGREDVLKEAVANKGPVSVGVDARHPSFFLYRSGVYYEPSCTQNVNHGVLVVGYGDLNGKEYWLVKNSWGHNFGEEGYIRMARNKGNHCGIASFPSYPEI SEQ ID NO: 44: Human AEP amino acid sequence (UniProt accession number Q99538) MVWKVAVFLSVALGIGAVPIDDPEDGGKHWVVIVAGSNGWYNYRHQADACHAYQIIHRNGIPDEQIVVMMYDDIAYSEDNPTPGIVINRPNGTDVYQGVPKDYTGEDV TPQNFLAVLRGDAEAVKGIGSGKVLKSGPQDHVFIYFTDHGSTGILVFPNEDLHVKDLNETIHYMYKHKMYRKMVFYIEACESGSMMNHLPDNINVYATTAANPRESS YACYYDEKRSTYLGDWYSVNWMEDSDVEDLTKETLHKQYHLVKSHTNTSHVMQYGNKTISTMKVMQFQGMKRKASSPVPLPPVTHLDLTPSPDVPLTIMKRKLMNTND LEESRQLTEEIQRHLDARHLIEKSVRKIVSLLAASEAEVEQLLSERAPLTGHSCYPEALLHFRTHCFNWHSPTYEYALRHLYVLVNLCEKPYPLHRIKLSMDHVCLGHY SEQ ID NO: 45 (BoNT / A1: UniProt P10845) [ka] In some embodiments, valine 27 can be substituted with alanine, as shown in SEQ ID NO:117. The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 46 (BoNT / A2: GenBank accession number X73423.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 47 (BoNT / A3: GenBank accession number DQ185900.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 48 (BoNT / A4: GenBank accession number EU341307.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 49 (BoNT / A5: GenBank accession number EU679004.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 50 (BoNT / A6: GenBank accession number FJ981696.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 51 (BoNT / A7: GenBank accession number JQ954969.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 52 (BoNT / A8: GenBank accession number KM233166.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 53 (BoNT / B1: UniProt P10844) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 54 (BoNT / B2: GenBank accession number AB084152.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 55 (BoNT / B3: GenBank accession number EF028400.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 56 (BoNT / B4: GenBank accession number EF051570.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 57 (BoNT / B5: GenBank accession number EF033130.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 58 (BoNT / B6: GenBank accession number AB302852.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 59 (BoNT / B7: GenBank accession number JQ354985.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 60 (BoNT / B8: GenBank accession number JQ964806.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 61 (BoNT / C1: UniProt P18640) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 62 (BoNT / D: UniProt P19321) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 63 (BoNT / CD: GenBank accession number AB200360.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 64 (BoNT / DC: GenBank accession number AB745660.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 65 (BoNT / E: UniProt Q00496) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 66 (BoNT / E1: GenBank accession number GQ244314.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 67 (BoNT / E2: GenBank accession number EF028404.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 68 (BoNT / E3: GenBank accession number EF028403.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 69 (BoNT / E4: GenBank accession number AB088207.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 70 (BoNT / E5: GenBank accession number AB037711.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 71 (BoNT / E6: GenBank accession number AM695759.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 72 (BoNT / E7: GenBank accession number JN695729.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 73 (BoNT / E8: GenBank accession number JN695730.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 74 (BoNT / E9: GenBank accession number JX424534.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 75 (BoNT / E10: GenBank accession number KF861917.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 76 (BoNT / E11: GenBank accession number KF861875.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 77 (BoNT / E12: GenBank accession number KM370319.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 78 (BoNT / F1: UniProt A7GBG3) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 79 (BoNT / F2: GenBank accession number GU213209.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 80 (BoNT / F3: GenBank accession number GU213227.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 81 (BoNT / F4: GenBank accession number GU213214.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 82 (BoNT / F5: GenBank accession number GU213211.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 83 (BoNT / F6: GenBank accession number M92906.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 84 (BoNT / F7: GenBank accession number GU213233.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 85 (BoNT / G: UniProt Q60393) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 86 (BoNT / FA: GenBank accession number KGO15617.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 87 (Polypeptide sequence of BoNT / X) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 88 (TeNT: UniProt P04958) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 89 (BoNT / D activation loop) CLRLTKNSRDDSTC SEQ ID NO: 90 (BoNT / DC activation loop) CLRLTRNSRDDSTC SEQ ID NO: 91 (BoNT / C1 and CD activation loop) CHKAIDGRSLYNKTLDC SEQ ID NO: 92 (BoNT / A4 activation loop) CVRGIITSKTKSLDEGYNKALNELC SEQ ID NO: 93 (BoNT / A5 and A7 activation loop) CVRGIITSKTKSLDEGYNKALNDLC SEQ ID NO: 94 (BoNT / A1 and A6 activation loop) CVRGIITSKTKSLDKGYNKALNDLC SEQ ID NO: 95 (BoNT / A3 activation loop) CVRGIIPFKTKSLDEGYNKALNYLC SEQ ID NO: 96 (BoNT / A2 and A8 activation loop) CVRGIIPFKTKSLDEGYNKALNDLC SEQ ID NO: 97 (BoNT / H activation loop) CSNSNTKNSLC SEQ ID NO: 98 (BoNT / E1 to E5, E9, and E12 activation loops) CKNIVSVKGIRKSIC SEQ ID NO: 99 (BoNT / E11 activation loop) CTNIFSPKGIRKSIC SEQ ID NO: 100 (BoNT / E7, E8, and E10 activation loop) CKNIVFSKGITKSIC SEQ ID NO: 101 (BoNT / E6 activation loop) CKNIVFSKGIRKSIC SEQ ID NO: 102 (BoNT / F7 activation loop) CKSIVSKKGTKNSLC SEQ ID NO: 103 (BoNT / F5 activation loop) CLNSSFKKNTKKPLC SEQ ID NO: 104 (BoNT / F1 and F6 activation loop) CKSVIPRKGTKAPPRLC SEQ ID NO: 105 (BoNT / F4 activation loop) CKSIIPRKGTKAPPRLC SEQ ID NO: 106 (BoNT / F2 and F3 activation loop) CKSIIPRKGTKQSPSLC SEQ ID NO: 107 (TeNT activation loop) CKKIIPPTNIRENLYNRTASLTDLGGELC SEQ ID NO: 108 (BoNT / G activation loop) CKPVMYKNTGKSEQC SEQ ID NO: 109 (BoNT / B4 activation loop) CKSVKVPGIC SEQ ID NO: 110 (BoNT / B2, B3, B6, and B8 activation loop) CKSVRAPGIC SEQ ID NO: 111 (BoNT / B1, B5, and B7 activation loop) CKSVKAPGIC SEQ ID NO: 112 (BoNT / X activation loop) CPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGC SEQ ID NO: 113 Metal-coordinated SNARE cleavage motif HEXXH SEQ ID NO: 114 (TEV cleavage site) ENLYFQG SEQ ID NO: 115 (thrombin cleavage site) LVPRGS SEQ ID NO: 116 (PreScission cleavage site) LEVLFQGP SEQ ID NO: 117 (BoNT / A GenBank accession number AF488749.1) [ka] The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 118: Unmodified BoNT / AB chimera [ka] Syt II binding mutations E1191M and S1199Y are shown in bold and underlined. The intrinsic activation loop is indicated by an underlined dash. SEQ ID NO: 119: LC / A1-Cloop-H N / A1 [ka] LC / A1 is shown in italics. The BoNT / C activation loop is shown in bold and underlined. H N / A1 is C-terminal to the C activation loop and is not underlined or italicized. SEQ ID NO: 120: LC / X-Cloop-H N / X [ka] LC / X is shown in italics. The BoNT / C activation loop is shown in bold and underlined. H N / X is C-terminal to the C activation loop and is not underlined or italicized. The 2xAP linker is indicated by an underlined dash. SEQ ID NO: 121: LC / X-EndoSite-H N / X [ka] LC / X is shown in italics. The EndoSite activation loop is shown in bold and underlined. H N / X is C-terminal to the C activation loop and is not underlined or italicized. The 2xAP linker is indicated by an underlined dash. SEQ ID NO: 122 (additional protease cleavage site) xDxxxLL x is any amino acid. SEQ ID NO: 123 (additional protease cleavage site) xExxxLL x is any amino acid. SEQ ID NO: 124 (additional protease cleavage site) xExxxIL x is any amino acid. SEQ ID NO: 125 (additional protease cleavage site) xExxxLM x is any amino acid. SEQ ID NO: 126 (Influenza virus hemagglutinin translocation domain) GLFGAIAGFIENGWEGMIDGWYG SEQ ID NO: 127 (Exemplary Endosite Extrinsic Activation Loop) [ka] AEP cleavage sites (QEAANERQQ, PDLKNVKS, and SGLTNIKTE) are shown in bold. The CathB cleavage sites (DLFGFVGL and GFVGLFRG) are double underlined. The CathL cleavage sites (QAKKDFFSSHPLREPVNATED, ELVTPARD, RDFGHFGL, and GLFRGHHP) are indicated by dotted underlines. SEQ ID NO: 128 (Exemplary Endosite Extrinsic Activation Loop) [ka] AEP cleavage sites (QLGKNEEG and SGLTNIKTE) are shown in bold. The CathB cleavage sites (GLFGFVGL and GFVGLFRG) are double underlined. The CathL cleavage sites (GLFRGHHP, ELVTPARD, and RDFGHFGL) are indicated by dotted underlines. SEQ ID NO: 129 (Exemplary Endosite Extrinsic Activation Loop) [ka] The AEP cleavage sites (PGGGNKKIE and PDLKNVKSK) are shown in bold. The CathB cleavage sites (DLFGFVGL and GFVGLFRG) are double underlined. The CathL cleavage sites (GLFRGHHP and ELVTPARD) are indicated by dotted underlines. SEQ ID NO: 130 (Exemplary Endosomal Protease (AEP) Cleavage Site) PDLKNVKS SEQ ID NO: 131 (Exemplary endosomal protease (CathB) cleavage site) DLFGFVGL SEQ ID NO: 132 (Exemplary endosomal protease (CathB) cleavage site) GFVGLFRG SEQ ID NO: 133 (Exemplary endosomal protease (CathB) cleavage site) GSGLFGFVGGSG SEQ ID NO: 134 (Exemplary endosomal protease (CathB) cleavage site) LFGFVGLFGFVG SEQ ID NO: 135 (Exemplary endosomal protease (CathB) cleavage site) LFGFVGLFGFVGLFGFVG SEQ ID NO: 136 (Exemplary endosomal protease (CathB) cleavage site) GLFGFVGL SEQ ID NO: 137 (Exemplary endosomal protease (CathL) cleavage site) QAKKDFFSSHPLREPVNATED SEQ ID NO: 138 (Exemplary endosomal protease (CathL) cleavage site) ELVTPARD SEQ ID NO: 139 (Exemplary endosomal protease (CathL) cleavage site) RDFGHFGL SEQ ID NO: 140 (Exemplary endosomal protease (CathL) cleavage site) GLFRGHHP SEQ ID NO: 141 (Exemplary endosomal protease (CathL) cleavage site) GSGLFRGHHPDGSG SEQ ID NO: 142 (Exemplary endosomal protease (CathL) cleavage site) LFRGHHPDLFRGHHPD SEQ ID NO: 143 (Exemplary endosomal protease (CathL) cleavage site) ELVTPARDFGHFGLS SEQ ID NO: 144 (Exemplary endosomal protease (CathL) cleavage site) QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFLGTNE SEQ ID NO: 145 (Exemplary endosomal protease (CathL) cleavage site) LFRGHHPDSTSQKSIVAYTMSLGADSS SEQ ID NO: 146 (Exemplary endosomal protease (CathL) cleavage site) STSQKSIVAYTMSLGADSSLFRGHHPD SEQ ID NO: 147 (Exemplary endosomal protease (CathL) cleavage site) STSQKSIVAYTMSLGADSSSTSQKSIVAYTMSLGADSS SEQ ID NO: 148 (Exemplary endosomal protease (CathL) cleavage site) LFRGHHPDLFRGHHPDLFRGHHPD SEQ ID NO: 149 (Exemplary endosomal protease (CathL) cleavage site) ELVTPARDFGHFGLSELVTPARDFGHFGLS SEQ ID NO: 150 (Exemplary endosomal protease (CathL) cleavage site) STSQKSIVAYTMSLGADSSELVTPARDFGHFGLSLFRGHHPD SEQ ID NO: 151 (Exemplary endosomal protease (AEP) cleavage site) QLGKNEEG SEQ ID NO: 152 (Exemplary endosomal protease (CathD) cleavage site) GERGFFYTPKT SEQ ID NO: 153: GS spacer consensus sequence (Gly-Gly-Gly-Gly-Ser) n SEQ ID NO: 154: GS5 spacer GGGGS SEQ ID NO: 155: GS10 spacer GGGGSGGGGS SEQ ID NO: 156: GS15 spacer GGGGSGGGGSGGGGS SEQ ID NO: 157: GS20 spacer GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 158: GS25 spacer GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 159: LH N / A1-H C Exemplary Modifications of B1 Chimera [ka] LC / A is shown in italics. The EndoSite activation loop is shown in bold and underlined. H N / A is C-terminal to the EndoSite activation loop and is not underlined or italicized. SEQ ID NO: 160: Exemplary LC / X-EndoSite-H N / X [ka] LC / X is shown in italics. The EndoSite activation loop is shown in bold and underlined. H N / X is C-terminal to the C activation loop and is not underlined or italicized. The 2xAP linker is indicated by an underlined dash. SEQ ID NO: 161: Exemplary LC / X-EndoSite-H N / X [ka] LC / X is shown in italics. The EndoSite activation loop is shown in bold and underlined. H N / X is C-terminal to the C activation loop and is not underlined or italicized. The 2xAP linker is indicated by an underlined dash. SEQ ID NO: 162: Exemplary LC / X-EndoSite-H N / X [ka] LC / X is shown in italics. The EndoSite activation loop is shown in bold and underlined. H N / X is C-terminal to the C activation loop and is not underlined or italicized. The 2xAP linker is indicated by an underlined dash. SEQ ID NO: 163: LC / X-Cloop-H N Exemplary Nucleic Acids Encoding / X [ka] The nucleic acid encoding Cloop is shown in bold and underlined. The nucleic acid encoding the 2xAP linker is indicated by an underlined dash. SEQ ID NO: 164: Exemplary Nucleic Acid Cloop tgtcataaagccattgatggtcgcagcctgtataacaaaaccctggat SEQ ID NO: 165: An exemplary nucleic acid encoding the exogenous activation loop of SEQ ID NO: 127 [ka] SEQ ID NO: 166: An exemplary nucleic acid encoding the exogenous activation loop of SEQ ID NO: 128 [ka] SEQ ID NO: 167: An exemplary nucleic acid encoding the exogenous activation loop of SEQ ID NO: 129 [ka] SEQ ID NO: 168: An exemplary nucleic acid encoding a modified retargeted BoNT / X comprising the exogenous activation loop of SEQ ID NO: 127 [ka] The nucleic acid encoding Cloop is shown in bold and underlined. The nucleic acid encoding the 2xAP linker is indicated by an underlined dash. SEQ ID NO: 169: An exemplary nucleic acid encoding a modified retargeted BoNT / X comprising the exogenous activation loop of SEQ ID NO: 128 [ka] The nucleic acid encoding Cloop is shown in bold and underlined. The nucleic acid encoding the 2xAP linker is indicated by an underlined dash. SEQ ID NO: 170: An exemplary nucleic acid encoding a modified retargeted BoNT / X comprising the exogenous activation loop of SEQ ID NO: 129 [ka] The nucleic acid encoding Cloop is shown in bold and underlined. The nucleic acid encoding the 2xAP linker is indicated by an underlined dash. SEQ ID NO: 171: Exemplary cathepsin L cleavage site [ka] SEQ ID NO: 172: Exemplary cathepsin L cleavage site [ka] SEQ ID NO: 173: Exemplary cathepsin L cleavage site [ka] SEQ ID NO: 174: Exemplary cathepsin L cleavage site QAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNEP SEQ ID NO: 175: Exemplary cathepsin L cleavage site QAKKDFFSSHPL SEQ ID NO: 176: Exemplary cathepsin L cleavage site REPVNATEDPSSGYYS SEQ ID NO: 177: Exemplary cathepsin L cleavage site TTIRYQATGFGTNE SEQ ID NO: 178: Exemplary cathepsin L cleavage site [ka] SEQ ID NO: 179: Exemplary cathepsin L cleavage site EVDLLIGSS SEQ ID NO: 180: Exemplary cathepsin L cleavage site EVDLLIGSSGE SEQ ID NO: 181: Exemplary cathepsin B cleavage site GLAGFLGG SEQ ID NO: 182: Exemplary cathepsin B cleavage site GLFGFVGG SEQ ID NO: 183: Exemplary cathepsin B cleavage site TVGSFGFE SEQ ID NO: 184: Exemplary cathepsin B cleavage site TVGSFGFEGG SEQ ID NO: 185: Exemplary cathepsin D cleavage site LASLLELPEFLLFLQ SEQ ID NO: 186: Exemplary cathepsin D cleavage site GLTTELFSPVD SEQ ID NO: 187: Exemplary AEP cleavage site LERNSNLVGAA SEQ ID NO: 188: Core cathepsin L cleavage motif MSLGADSS SEQ ID NO: 189: Core cathepsin L cleavage motif LFRGHHP SEQ ID NO: 190: Core cathepsin L cleavage motif GLFRGHHP SEQ ID NO: 191: Core cathepsin L cleavage motif KDFFSSHP SEQ ID NO: 192: Core cathepsin L cleavage motif EPVNATED SEQ ID NO: 193: Core cathepsin L cleavage motif TGFGTNE SEQ ID NO: 194: Core cathepsin L cleavage motif TGFGTNEP SEQ ID NO: 195: Core cathepsin L cleavage motif QKVGKAMY SEQ ID NO: 196: Core cathepsin L cleavage motif LLIGSS SEQ ID NO: 197: Core cathepsin L cleavage motif LLIGSSGE SEQ ID NO: 198: Core cathepsin B cleavage motif GSFGFE SEQ ID NO: 199: Core cathepsin B cleavage motif GSFGFEGG SEQ ID NO: 200: Core cathepsin D cleavage motif VEKLLELK SEQ ID NO: 201: Core cathepsin D cleavage motif VITLVMLK SEQ ID NO: 202: Core cathepsin D cleavage motif GMELIVSQ SEQ ID NO: 203: Core cathepsin D cleavage motif QPYLEMDL SEQ ID NO: 204: Core cathepsin D cleavage motif EYALLYKL SEQ ID NO: 205: Core cathepsin D cleavage motif LAEEEVVI SEQ ID NO: 206: Core cathepsin D cleavage motif LASLLELP SEQ ID NO: 207: Core cathepsin D cleavage motif TTELFSPV SEQ ID NO: 208: Core AEP cleavage motif EAANERQQ SEQ ID NO: 209: Core AEP cleavage motif GLTNIKTE SEQ ID NO: 210: Core AEP cleavage motif DLKNVKSK SEQ ID NO: 211: Core AEP cleavage motif GGGNKKIE SEQ ID NO: 212: Core AEP cleavage motif LGKNEEGA SEQ ID NO: 213: Core AEP cleavage motif ERNSNLV SEQ ID NO: 214: Core AEP cleavage motif LERNSNLV [Example]

[0322] The present invention is further illustrated by the following examples, which are intended to be purely illustrative of the present invention and are in no way limiting. In addition to the data presented below, the contents of PCT / GB2022 / 050756, particularly the Examples section, are incorporated herein by reference. This example relates to a modified clostridial neurotoxin that contains a furin cleavage site rather than an endosomal protease cleavage site, but includes data obtained from modified clostridial neurotoxins that share features common to those of the present invention, and related synthetic methods.

[0323] Example 1: Design and production of BoNTs modified to contain an endosomal protease cleavage site The modified BoNTs were produced by determining the desired amino acid sequence, back-translating it into the corresponding nucleic acid sequence, and then codon-optimizing it for recombinant expression in bacteria.

[0324] The resulting gene sequence was verified to ensure that commonly used restriction sites (NdeI, XhoI, BamHI, HindIII, NcoI, and EcoRI) were not present within the sequence. A start codon was added to the 5' end, a His tag (optionally cleavable) and a stop codon to the 3' end, and appropriate terminal restriction sites (e.g., an NdeI restriction site at the 5' end and a BamHI restriction site at the 3' end) were added to allow subcloning into expression vectors.

[0325] The gene sequences of the modified BoNTs were then subcloned into the pK8 vector (containing a kanamycin resistance gene, a T7 promoter, a T7 terminator, a pBR322 origin of replication, and a multiple cloning site). Plasmids of each modified BoNT were then amplified in E. coli DH5α under kanamycin selection and extracted by miniprep using standard molecular biology techniques.

[0326] The E. coli expression strain BL21 harboring λDE3 was then transformed with the plasmid DNA, spread onto agar supplemented with kanamycin, and incubated overnight at 37°C. Colonies were then picked and used to prepare glycerol stocks. A stab was then used to inoculate 100 mL of modified TB medium supplemented with kanamycin, which was then incubated overnight at 37°C with shaking at 225 RPM to provide aeration. Ten mL of this starter culture was used to inoculate several baffled conical flasks (each containing up to 1 L of the same nutrient medium and antibiotics). The cultures were grown under the same conditions for several hours to an optical density (A600) of 0.6 or greater. The incubator temperature was then set to 16°C. The cultures were then induced to express the modified BoNT by adding IPTG one hour later. After 20 hours, the cells were harvested by centrifugation and stored at -80°C until use.

[0327] Cells were thawed (5 mL / g cells) in 0.25 M NaCl in 50 mM Tris pH 7.4 and lysed at 4 °C by passing twice through a cell homogenizer at 20 k PSI or by sonication (10 × 30 sec on / off). Cell debris was removed by centrifugation, and the clarified supernatant was loaded onto a nickel affinity column (pre-equilibrated with 0.5 M NaCl in 50 mM Tris pH 7.4 ("Buffer NA") using an FPLC system (GE). The column was washed with Buffer NA until a stable baseline at A280 was achieved. The washed and eluted protein was collected from the column using a linear gradient of 0 to 0.5 M imidazole in Buffer NA over 25 column volumes (CV), while 3 mL fractions were collected.

[0328] All collected material was stored at 4°C while samples were analyzed by SDS-PAGE (Invitrogen) with staining. Based on protein markers, fractions showing a strong protein band with the calculated MW of the target molecule were pooled, and total protein concentration was measured using a Nanodrop (Thermo Fisher).

[0329] Pooled fractions were desalted into 50 mM Tris pH 8 ("Buffer QA") and further purified by anion exchange chromatography (e.g., Q HP). After washing the column with Buffer QA until a stable baseline was reached, the protein was eluted with a linear gradient of 0 to 0.5 M NaCl in Buffer QA over 25 CV. Samples were analyzed by SDS-PAGE, and fractions containing pure target were desalted into 150 mM NaCl in 50 mM HEPES pH 7.2, then aliquoted and stored at -80°C. Samples of the final product in the presence and absence of DTT were analyzed by SDS-PAGE, revealing a single band.

[0330] Using the above method, structure LC / A1-Cloop-H N Three modified BoNTs with the same targeting moiety (TM), BoNT / A1 light chain (LC / A1), and BoNT / A1 translocation domain (H) were produced (EndoSite=CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945)). All three modified BoNTs share the same targeting moiety (TM), BoNT / A1 light chain (LC / A1), and BoNT / A1 translocation domain (H). N / A1), but the endosomal protease cleavage sites and their combinations are different. N The / A1 sequence is SEQ ID NO: 119. BIO4934 contains the endosomal protease cleavage site of SEQ ID NO: 35, which itself contains one cleavage site for cathepsin D and two cleavage sites for cathepsin L. BIO4935 contains the endosomal protease cleavage site of SEQ ID NO: 36, which itself contains two cleavage sites for cathepsin L. BIO4945 contains the endosomal protease cleavage site of SEQ ID NO: 37, which itself contains one cleavage site for cathepsin B and two cleavage sites for cathepsin L.

[0331] As pH controls, samples of CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945) were incubated in 1 mM DTT with either PBS at pH 7.2 or 50 mM MES at pH 5 for 3 hours at room temperature without the corresponding endosomal protease and then reduced for analysis.

[0332] As shown in Figure 1A, when run on a Coomassie gel, CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945) all had fainter bands visible at pH 7.2 compared to pH 5, corresponding to the full-length modified BoNT. N The / A1-EndoSite-TM-His6 bands were similar in intensity at both pHs. Faint bands at >20 kDa were observed in all CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945), indicating impurities. Importantly, no bands at approximately 15 kDa were visible (at pH 7.2 or pH 5), indicating that cleavage by EndoSite in the absence of the corresponding endosomal proteases was zero / below the limit of detection / negligible by protein detection.

[0333] Western blot analysis was also performed using anti-LC / A1 antibody (Fig. 1B) and anti-His tag antibody (data not shown). As shown in Fig. 1B, when anti-LC / A1 antibody was used, full-length bands, LC / A1 bands, and H bands were detected at two pH levels. N No difference in signal intensity was observed for the A1-EndoSite-TM-His6 band. Faint bands were observed at approximately 30 kDa and 20 kDa, as well as at >50 kDa. This may be due to the presence of impurities and / or H NThis suggests cross-reactivity with the / A1-EndoSite-TM-His6 strand (possibly as an artifact of Western blot overexposure). Nevertheless, no band at approximately 15 kDa was visible (negligible, undetectable, or undetectable) at EndoSite in the absence of the corresponding endosomal protease.

[0334] Using an anti-His tag antibody, it was again confirmed that buffer pH had no significant effect on CatDReo (BIO4934), Ebo (BIO4935), and CatBL (BIO4945). Several faint bands were observed at <20 kDa. These are hypothesized to be artifactual cross-reactivity or impurities, or trace cleavage products that could only be detected by overexposed Western blots.

[0335] Example 2: BoNTs modified to contain a cathepsin L1 cleavage site are efficiently cleaved by cathepsin L1 The three modified BoNTs (LC / A1-Cloop-H) produced in Example 1 N / A1-EndoSite-TM-His6 (EndoSite = CatDReo (BIO4934), Ebo (BIO4935), or CatBL (BIO4945))) were examined.

[0336] The ability of cathepsin L to cleave BIO4934, BIO4935, and BIO4945 was tested by incubating 90 μg / mL of each modified BoNT with serial dilutions of cathepsin L1 in 50 mM MES pH 5 reduced with DTT for 2 hours at room temperature and resolving by SDS PAGE for Coomassie staining and Western blot analysis.

[0337] BIO4934, BIO4935, and BIO4945 are each approximately 118 kDa (approximately 100 kDa LH N / A+ consists of approximately 18 kDa TM-HT).

[0338] BIO4934, BIO4935, and BIO4945 are all sensitive to cathepsin L, and are cleaved to approximately 50 kDa (LC / A and H). N A doublet of a band at 1 kDa ( / A, upon reduction) and a >15 kDa TM-HT band was obtained. Cleavage of each of the BIO4934, BIO4935, and BIO4945 modified BoNTs was concentration-dependent, with even low concentrations of cathepsin L1 (14 ng / mL) achieving some degree of cleavage. Exemplary data for BIO4934 and BIO4935 are shown in Figures 2A and 2B, respectively.

[0339] Therefore, these data demonstrate that cathepsin L can be successfully used to cleave and activate the BIO4934, BIO4935, and BIO4945 modified BoNTs.

[0340] Example 3: BoNTs modified to contain a cathepsin B cleavage site are efficiently cleaved by cathepsin B The CatBL (BIO4945) modified BoNT produced in Example 1 was examined for its sensitivity to cathepsin B.

[0341] The ability of cathepsin B to cleave BIO4945 was tested by incubating 0.3 mg / mL CatBL (BIO4945) with serial dilutions of cathepsin B for 2 hours at room temperature in 50 mM MES pH 5 reduced with DTT and resolved by SDS PAGE for Coomassie staining and Western blot analysis.

[0342] As shown in Figure 3, BIO4945 is sensitive to cathepsin B, and is cleaved to a protein of approximately 50 kDa (LC / A and H). NA doublet of a band at 1 kDa ( / A, upon reduction) and a TM-HT band at >15 kDa is obtained. Cleavage of the BIO4945-modified BoNT was concentration-dependent, and even low concentrations of cathepsin B (5 ng / mL) achieved some degree of cleavage. Therefore, these data indicate that cathepsin B can be successfully used to cleave and activate the BIO4935-modified BoNT.

[0343] Example 4: BoNTs modified to contain an asparaginyl endopeptidase cleavage site are efficiently cleaved by asparaginyl endopeptidase The same TM, LC / A1, and H as described in Examples 1-3 were used. N / A1, but with an AEP cleavage site (LC / A1-Cloop-H N / A1-EndoSite-TM-His6(EndoSite=AEP(BIO4938))) was produced and tested. BIO4938 contains the endosomal protease cleavage site of SEQ ID NO: 38, which itself contains five cleavage sites for AEP.

[0344] BIO4938 at 150 μg / mL was incubated with serial dilutions of AEP for approximately 2 hours at room temperature in 50 mM MES pH 5 and reduced for analysis.

[0345] BIO4938 is approximately 119 kDa (approximately 100 kDa LH N / A+ consists of approximately 19 kDa AEP-TM-HT).

[0346] As shown in Figure 4, BIO4938 is sensitive to AEP and undergoes cleavage to produce approximately 50 kDa (LC / A and H). N A doublet of a band at 1 kDa ( / A, when reduced) and a TM-HT band at >15 kDa is obtained. Cleavage of the BIO4938-modified BoNT was concentration-dependent, and even low concentrations of AEP B (7 ng / mL) achieved some degree of cleavage.

[0347] Example 5: Design and production of retargeted BoNT / X with an endogenous activation loop modified to contain an endosomal protease cleavage site The method of Example 1 is repeated to produce a modified BoNT derived from the retargeted BoNT / X molecule. The modified BoNT / X has the structure LC / X-Cloop-H N / X-2xAP Linker-derived from retargeted BoNT / X with TM-tag (LC / X-Cloop-H N The / X-2xAP linker has the sequence set forth in SEQ ID NO: 120, and the tag is a cleavable affinity purification tag (e.g., His-tag). Alternatively, the modified BoNT / X can have the structural tag-LC / X-Cloop-H N The modified retargeted BoNT / X is derived from a retargeted BoNT / X having an N-terminal tag with a LC / X-2xAP linker-TM. The tag is optionally cleavable. The BoNT / C activation loop is replaced by an EndoSite activation loop (e.g., SEQ ID NOS: 127-129). Thus, the modified retargeted BoNT / X has the structure: LC / X-EndoSite-H N / X-2xAP Linker-TM-Tag or Tag-LC / X-EndoSite-H N / X-2xAP Linker-TM, LC / X-EndoSite-H N The LC / X-2xAP linker has the sequence shown in SEQ ID NOs: 160 to 162. N / X-2xAP is sensitive to one or more of AEP, cathepsin B, and / or cathepsin L.

[0348] LC / X-Cloop-H at set concentration N / X-2xAP Linker-TM-Tag or Tag-LC / X-EndoSite-H N / X-2xAP Linker-TM is treated with serial dilutions of AEP, cathepsin B, or cathepsin L and incubated for 2 hours at room temperature. Samples are reduced with DTT and analyzed by SDS-PAGE and Western blot (anti-tag) to assess EndoSite cleavage by AEP, cathepsin B, or cathepsin L compared to untreated samples. Protein gels show a decrease in intensity of a single band representing the single strand and the appearance of two smaller bands corresponding to the active duplex, with a concomitant increase in intensity. Anti-tag blots show similar results, except that the smaller of the two bands (due to untagged LC) is missing.

[0349] Target cells (e.g., primary cortical neurons) were cultured in triplicate wells on LC / X-Cloop-H plates. N / X-2xAP Linker-TM-Tag or Tag-LC / X-EndoSite-H N Cells are treated with serial dilutions of LC / X-2xAP Linker-TM and incubated for 24 hours. Cells are then harvested and lysed with 1x NuPAGE buffer, DTT, and benzonase. Lysates are then analyzed by Western blot for substrate cleavage by LC / X (e.g., VAMP2, VAMP4, or Ykt6) by measuring the disappearance of the substrate band and the appearance of cleaved fragment bands by densitometry. The amount of cleaved substrate is expressed as a percentage of the total uncleaved and cleaved substrate, and the concentration of target molecule required to induce half-maximal cleavage of the substrate (EC50) is calculated by nonlinear regression. This is compared with the concentration of the corresponding molecule without EndoSite (e.g., Cloop, LC / X-Cloop-H). N / X-2xAP Linker-TM-Tag or native Xloop, LC / X-Xloop-H N / X-2xAP Linker-TM-Tag, or Tag-LC / X-Xloop-H N / X-2xAP linker-TM).

[0350] Single-strand LC / X-EndoSite-H N / X-2xAP Linker-TM-Tag or Tag-LC / X-EndoSite-H N The LC / X-2xAP linker-TM exhibits substrate cleavage, while the corresponding single-stranded non-EndoSite retargeting BoNT / X (LC / X-Cloop-H) N / X-2xAP Linker-TM-Tag, Tag-LC / X-Cloop-H N / X-2xAP Linker-TM, LC / X-Xloop-H N / X-2xAP Linker-TM-Tag, or Tag-LC / X-Xloop-H N / X-2xAP Linker-TM) show no / minimal cleavage.

[0351] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the 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.

Claims

1. 1. A modified Clostridial neurotoxin comprising an endosomal protease cleavage site, wherein cleavage at said cleavage site results in the production of a di-chain form of said modified Clostridial neurotoxin.

2. The endosomal protease cleavage site is (a) asparagine endopeptidase (AEP), or (b) a cathepsin, optionally cathepsin L1, B, D, K, or S The modified Clostridial neurotoxin of claim 1, wherein the cleavage site is specific for .

3. The endosomal protease cleavage site is (a) an AEP core motif selected from SEQ ID NOs: 208, 209, 210, 211, 212, 213, and / or 214; (b) a cathepsin L core motif selected from SEQ ID NOs: 138, 188, 189, 190, 191, 192, 193, 194, 195, 196, and / or 197; (c) a cathepsin B core motif selected from SEQ ID NOs: 20, 181, 198, and / or 199, and / or (d) a cathepsin D core motif selected from SEQ ID NOs: 200, 201, 202, 203, 204, 205, 206, and / or 207.

3. The modified Clostridial neurotoxin of claim 1 or 2, comprising or consisting of:

4. 10. A modified Clostridial neurotoxin according to any one of the preceding claims, comprising: (a) the endosomal protease cleavage site is selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 41, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, and / or 187; (b) the endosomal protease cleavage site is a cathepsin L cleavage site comprising or consisting of one or more of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 27, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 171, 172, 173, 174, 175, 176, 177, 178, 179, and / or 180; (c) the endosomal protease cleavage site is a cathepsin B cleavage site comprising or consisting of one or more of SEQ ID NOs: 20, 28, 131, 132, 133, 134, 135, 136, 181, 182, 183, and / or 184; (d) the endosomal protease cleavage site is a cathepsin D cleavage site comprising or consisting of one or more of SEQ ID NOs: 21, 29, 30, 31, 32, 33, 152, 185, and / or 186; and / or (e) the modified Clostridial neurotoxin, wherein the endosomal protease cleavage site is an AEP cleavage site comprising or consisting of one or more of SEQ ID NOs: 22, 23, 24, 25, 26, 34, 130, 151, and / or 187.

5. 10. The modified Clostridial neurotoxin of any one of the preceding claims, wherein the modified Clostridial neurotoxin comprises an extrinsic activation loop comprising or consisting of any one of SEQ ID NOs: 35, 36, 37, 38, 127, 128, and / or 129.

6. 10. The modified Clostridial neurotoxin according to any one of the preceding claims, wherein the endogenous activation loop of the Clostridial neurotoxin, or a portion thereof, is replaced by an endosomal protease cleavage site.

7. 7. The modified Clostridial neurotoxin of claim 6, wherein the endogenous neurotoxin activation loop is one or more selected from SEQ ID NOs: 89-112.

8. The clostridial neurotoxin (a) botulinum neurotoxin (BoNT) serotype A, serotype B, serotype C, serotype D, serotype E, serotype F, serotype G, or serotype X, or tetanus neurotoxin (TeNT), or (b) chimeric or hybrid BoNTs; 10. The modified Clostridial neurotoxin of any one of the preceding claims, wherein

9. 9. The modified Clostridial neurotoxin of claim 8, (a) BoNT / A, optionally BoNT / A1, or (b) BoNT / X The modified Clostridial neurotoxin,

10. 10. A modified Clostridial neurotoxin according to any one of the preceding claims, comprising: (a) a single-chain clostridial neurotoxin encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, in which SEQ ID NO: 164 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site, optionally selected from SEQ ID NOs: 165, 166, and 167; and / or (b) a single-chain Clostridial neurotoxin comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 120, in which SEQ ID NO: 91 is replaced by at least one endosomal protease cleavage site or extrinsic activation loop, optionally selected from SEQ ID NOs: 127-129; The modified Clostridial neurotoxin.

11. Endogenous H of Clostridial Neurotoxins Ｃ or H ＣＣ 10. The modified Clostridial neurotoxin of any one of the preceding claims, which is a retargeted Clostridial neurotoxin, wherein is replaced by an exogenous targeting moiety (TM).

12. A modified Clostridial neurotoxin containing an endosomal protease cleavage site, comprising a polypeptide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity to SEQ ID NOs: 160-162.

13. 13. A method for proteolytically cleaving the modified Clostridial neurotoxin of any one of claims 1 to 12 into the corresponding di-chain Clostridial neurotoxin, the method comprising contacting the modified Clostridial neurotoxin with an endosomal protease specific for the endosomal protease cleavage site to produce the di-chain Clostridial neurotoxin.

14. A double-chain Clostridial neurotoxin obtainable by the method of claim 13.

15. A polynucleotide encoding a modified Clostridial neurotoxin as defined in any one of claims 1 to 11 or according to claim 12.

16. 16. An expression vector comprising the polynucleotide defined in claim 15 operably linked to a promoter.

17. 17. The polynucleotide of claim 15 or the expression vector of claim 16, (a) comprising or consisting of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, in which SEQ ID NO: 164 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site, optionally selected from SEQ ID NOs: 165, 166, and 167; and / or (b) encoding a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is replaced by at least one endosomal protease cleavage site or extrinsic activation loop, optionally selected from SEQ ID NOs: 127-129; The polynucleotide or the expression vector.

18. 13. A method for producing a modified Clostridial neurotoxin as defined in any one of claims 1 to 12, comprising the steps of expressing a polynucleotide as defined in claim 15 or 17 or an expression vector as defined in claim 16 or 17 in a cell and recovering the expressed modified Clostridial neurotoxin.

19. 19. The method of claim 18, further comprising the step of introducing into said cell a polynucleotide as defined in claim 15 or 17 or an expression vector as defined in claim 16 or 17.

20. A cell expressing a modified Clostridial neurotoxin as defined in any one of claims 1 to 12.

21. 21. A cell according to claim 20, comprising a polynucleotide as defined in claim 15 or 17 or an expression vector as defined in claim 16 or 17.

22. 15. A pharmaceutical composition comprising a modified clostridial neurotoxin as defined in any one of claims 1 to 12 or a two-chain clostridial neurotoxin as defined in claim 14, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, propellant, and / or salt.

23. 22. A modified Clostridial neurotoxin as defined in any one of claims 1 to 12, a double-chain Clostridial neurotoxin as defined in claim 14, or a pharmaceutical composition as defined in claim 22 for use in a method for preventing or treating a disease or disorder in which a need for treatment with a botulinum neurotoxin is indicated, optionally wherein the disease or disorder is a condition associated with unwanted immune secretion, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, orthomandibular ... dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), cosmetic therapeutic (cosmetic) uses that benefit from cell / muscle weakening (via SNARE downregulation or inactivation), neuromuscular disorders or conditions of eye movement (e.g. conjugate strabismus, vertical strabismus, lateral rectus palsy, nystagmus, myopathy due to thyroid abnormalities), writer's cramp, teeth grinding, Wilson's disease, tremors, tics, segmental myoclonus, spasticity, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder abnormalities, animus, back spasticity, muscle spasms or rigidity (Charley horse), levator pelvis syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), forehead grooves, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders.

24. 22. Use of a modified Clostridial neurotoxin as defined in any one of claims 1 to 12, a double-chain Clostridial neurotoxin as defined in claim 14, or a pharmaceutical composition as defined in claim 22 in the manufacture of a medicament for preventing or treating a disease or disorder in which a need for treatment with a botulinum neurotoxin is indicated, optionally wherein the disease or disorder is selected from the group consisting of conditions associated with unwanted immune secretions, strabismus, blepharospasm, squint, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, ... dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), cosmetic therapeutic (cosmetic) uses that benefit from cell / muscle weakening (via SNARE downregulation or inactivation), neuromuscular disorders or conditions of eye movement (e.g. conjugate strabismus, vertical strabismus, lateral rectus palsy, nystagmus, myopathy due to thyroid abnormalities), writer's cramp, teeth grinding, Wilson's disease, tremors, tics, segmental myoclonus, spasticity, spasticity due to chronic multiple sclerosis, spasticity resulting in bladder abnormalities, animus, back spasticity, muscle spasms or rigidity (Charley's horse), levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremor, teeth grinding, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain due to muscle spasms), headache (e.g., tension headache or migraine), phantom pain (e.g., phantom limb pain), forehead grooves, skin wrinkles, cancer, uterine disorders, urogenital disorders, urogenital neuropathy, bladder pain syndrome, interstitial cystitis, chronic neurogenic inflammation, and smooth muscle disorders.

25. 25. The modified Clostridial neurotoxin or pharmaceutical composition for use according to claim 23, or the use of the modified Clostridial neurotoxin or pharmaceutical composition according to claim 24, wherein the Clostridial neurotoxin is administered to the subject in a single-chain form.

26. 26. A modified Clostridial neurotoxin or pharmaceutical composition for use as described in claim 23 or 25, or the use of a modified Clostridial neurotoxin or pharmaceutical composition as described in claim 24 or 25, wherein the Clostridial neurotoxin or pharmaceutical composition is substantially free of a two-chain form of the Clostridial neurotoxin.

27. 27. The modified Clostridial neurotoxin or pharmaceutical composition for use according to claim 23, 25 or 26, or the use of the modified Clostridial neurotoxin or pharmaceutical composition according to any one of claims 24 to 26, wherein the Clostridial neurotoxin or the pharmaceutical composition comprises less than 400 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 300 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 200 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 100 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin, or less than 50 pg of double-chain Clostridial neurotoxin per 100 ng of single-chain Clostridial neurotoxin.

28. 15. A cosmetic composition comprising a modified clostridial neurotoxin as defined in any one of claims 1 to 12 or a two-chain clostridial neurotoxin as defined in claim 14, and a cosmetically acceptable carrier, excipient, diluent, adjuvant, propellant, and / or salt.

29. 29. Use of a cosmetic composition as defined in claim 28 for preventing or alleviating a cosmetic indication in which the application of a botulinum neurotoxin is indicated.

30. 29. The cosmetic composition of claim 28, or the use of the cosmetic composition of claim 28, wherein the Clostridial neurotoxin is administered to the subject in a single-chain form.

31. 31. The use of a cosmetic composition according to claim 28 or 30, or a cosmetic composition according to claim 29 or 30, wherein the clostridial neurotoxin or the cosmetic composition is substantially free of the two-chain form of the clostridial neurotoxin.

32. 32. The cosmetic composition of claim 28, 30, or 31, or use of the cosmetic composition of any one of claims 29 to 31, wherein the clostridial neurotoxin or the cosmetic composition comprises less than 400 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 300 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 200 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 100 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin, or less than 50 pg of double-chain clostridial neurotoxin per 100 ng of single-chain clostridial neurotoxin.

33. 1. A method for proteolytically cleaving a single-chain Clostridial neurotoxin into the corresponding two-chain Clostridial neurotoxin, said method comprising: (a) providing a single-chain Clostridial neurotoxin; (b) contacting the single-chain Clostridial neurotoxin with an endosomal protease; the single-chain Clostridial neurotoxin having an activation loop comprising or consisting of a polypeptide sequence as defined in any one of claims 3 to 5; The method, wherein the endosomal protease hydrolyzes the peptide bond of the activation loop to produce a di-chain clostridial neurotoxin.

34. 34. The method of claim 33, wherein the activation loop comprises or consists of a polypeptide sequence as defined in claim 4 or 5.

35. the single-chain clostridial neurotoxin (a) a modified Clostridial neurotoxin as defined in any one of claims 1 to 10; (b) encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 163, in which SEQ ID NO: 164 is replaced by a nucleotide sequence encoding at least one endosomal protease cleavage site, optionally selected from SEQ ID NOs: 165, 166, and 167; and / or (c) comprising a polypeptide sequence having at least 70% sequence identity to one or more of SEQ ID NO: 120, wherein SEQ ID NO: 91 within SEQ ID NO: 120 is optionally replaced by at least one endosomal protease cleavage site or extrinsic activation loop selected from SEQ ID NOs: 127-129; 35. The method of claim 33 or 34.

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