EA041568B1 - NEW BOTULINIC NEUROTOXIN AND ITS DERIVATIVES - Google Patents

Description

Link to related applications

According to the application for this invention in accordance with 35 U.S.C. § 119(e) claims priority under U.S. Patent Application Serial No. 62/360239, filed July 8, 2016, which is incorporated herein by reference in its entirety.

Government support

The present invention was made with government support in accordance with R01NS080833 awarded by the National Institutes of Health. The government has certain rights to the present invention.

Background of the Invention

Clostridial botulinum neurotoxins (BoNT) are among the most dangerous bioterrorist potentials and are also being used clinically to treat an increasing list of medical conditions. There are seven BoNT serotypes (BoNT/A-G) currently known. In recent years, BoNT has been widely used to treat a growing list of medical conditions: local injections of small amounts of toxins can attenuate neuronal activity at targeted sites, which can be beneficial for many medical conditions, as well as for cosmetic purposes. As the use of BoNT expands, limitations and side effects are reported. The main limitation is the production of neutralizing antibodies in patients, which makes further treatment ineffective. Stopping BoNT use often leaves patients without other effective treatments/alleviation for their disorders. Side effects associated with the use of BoNT range from short-term, non-severe events such as ptosis and diplopia to life-threatening events, even death. The limitations and side effects of BoNT are largely dose related. There is considerable interest in the development of new types of BoNT as therapeutic toxins. Over the past 45 years, no new types of BoNT have been identified.

Brief summary of the present invention

The present disclosure is based at least in part on the identification of a novel BoNT serotype, BoNT/X, by searching a genomic database of Clostridium Botulinum strains. BoNT/X has the least sequence identity to other BoNTs and is not recognized by antisera raised against known types of BoNTs. BoNT/X cleaves SNARE proteins like other BoNTs. However, BoNT/X also cleaves some SNARE proteins that other BoNTs cannot cleave, such as VAMP4, VAMP5, and Ykt6. Compositions and methods for treating diseases using BoNT/X are presented. Also provided herein are methods for producing BoNT/X.

Therefore, in certain aspects of the present disclosure, isolated Clostridial botulinum neurotoxin (BoNT) polypeptides are provided comprising the amino acid sequence of SEQ ID NO: 1.

In some aspects of the present disclosure, isolated BoNT polypeptides are provided comprising an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to SEQ ID NO: 1. In some embodiments, the isolated BoNT polypeptide consists of the amino acid sequence under SEQ ID NO: 1.

In some aspects of the present disclosure, isolated BoNT polypeptides are provided comprising the amino acid sequence of SEQ ID NO: 2. In some aspects of the present disclosure, isolated BoNT polypeptides are provided, the amino acid sequence of which is characterized by at least 85%, at least 86%, at least 87 %, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with respect to SEQ ID NO: 2. In some embodiments, the isolated BoNT polypeptide consists of the amino acid sequence under SEQ ID NO: 2.

In some aspects of the present disclosure, isolated BoNT polypeptides are provided comprising the amino acid sequence of SEQ ID NO: 3. In some aspects of the present disclosure, isolated polypeptides are provided.

BoNTs whose amino acid sequence is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99 .5% identity with respect to SEQ ID NO: 3. In some embodiments, the isolated polypeptide

- 1 041568

BoNT consists of the amino acid sequence under SEQ ID NO: 3.

In some aspects of the present disclosure, modified BoNT polypeptides are provided consisting of one or more substitution mutations at the position corresponding to C461, C467, and C1240 in SEQ ID NO: 1. In some embodiments, the substitution mutation(s) corresponds to C461S , C461A, C467S, C467A, C1240S, C1240A, C461S/C1240S, C416S/C1240A, C461A/C1240S, C461A/C1240A, C467S/C1240S, C461S/C1240A, C467A/C1240S: NO.

In accordance with some embodiments, the modified BoNT polypeptide comprises the amino acid sequence of any one of SEQ ID NO ID NOs: 4-17. In some embodiments, the modified BoNT polypeptide contains an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to any sub

SEQ ID NO: 4-17, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified BoNT polypeptide consists of the amino acid sequence of any of SEQ ID NO: 4-17.

In some aspects of the present disclosure, modified BoNT polypeptides are provided having a single substitution type mutation at a position corresponding to C461 or C467 in SEQ ID NO: 2.

In some embodiments, the substitution mutation corresponds to C461S, C461A, C467S, C467A, C1240S, C1240A, C461S/C1240S, C416S/C1240A, C461A/C1240S, C461A/C1240A, C467S/C1240S, C460A/C1240A or C467A/C1240A in SEQ ID NO: 2.

In accordance with some embodiments, the modified BoNT polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 18-21. In some embodiments, the modified BoNT polypeptide contains an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to any sub

SEQ ID NO: 18-21, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified BoNT polypeptide consists of the amino acid sequence of any of SEQ ID NO: 18-21.

In some aspects of the present disclosure, chimeric BoNT polypeptides are provided comprising the amino acid sequence of any of SEQ ID NOs: 22-24. In some embodiments, the chimeric BoNT polypeptide comprises an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to any under SEQ ID NO: 22-24, while the polypeptide does not have the amino acid sequence under SEQ ID NO: 1 or SEQ ID NO: 2. In accordance with some embodiments, the chimeric BoNT polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 22-24.

In some embodiments, the chimeric BoNT polypeptide further comprises a single substitution mutation at the position corresponding to C461 or C467 in SEQ ID NO: 2.

In accordance with some embodiments, the chimeric BoNT polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 25-30. In some embodiments, the chimeric BoNT polypeptide comprises an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to any under SEQ ID NO: 25-30. In some embodiments, the chimeric BoNT polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 25-30.

In accordance with some embodiments, the BoNT polypeptide enters the cell. In some embodiments, the BoNT polypeptide cleaves the SNARE protein in the cell. In some embodiments, the SNARE protein is selected from the group consisting of SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

In some embodiments, the SNARE protein is

VAMP1. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 39.

In some embodiments, the SNARE protein is VAMP2. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 40.

In some embodiments, the SNARE protein is VAMP3. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO:41.

In some embodiments, the SNARE protein is VAMP4. In some embodiments, BoNT cleaves between amino acid residues corresponding to K87 and S88 of SEQ ID NO: 42.

In some embodiments, the SNARE protein is VAMP5. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R40 and S41 of SEQ ID NO:43.

In some embodiments, the SNARE protein is Ykt6. According to some embodiments, BoNT cleaves between amino acid residues corresponding to K173 and S174 of SEQ ID NO:44.

In some embodiments, the BoNT polypeptide has increased stability relative to its corresponding wild-type BoNT polypeptide.

In accordance with some embodiments, the cell is a secretory cell. In accordance with some embodiments, the cell is a neuronal cell. In some embodiments, the cell is an immune cell. In accordance with some embodiments, the BoNT polypeptide suppresses neuronal activity. In some embodiments, the BoNT polypeptide induces atrophic paralysis. In accordance with some embodiments, the cell is a cultured cell. In accordance with some embodiments, the cell is in vivo. In accordance with some embodiments, the cell is derived from a mammal. In some embodiments, the mammal is a human. In accordance with some embodiments, the mammal is a rodent. In accordance with some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat.

In some embodiments, the BoNT polypeptide does not cross-react with an antibody against serotype A, B, C, D, E, F, or GBoNT.

In other aspects of the present disclosure, nucleic acid molecules are provided, consisting of a polynucleotide encoding a polypeptide containing an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99%, or at least 99.5% or 100% identity with respect to the BoNT polypeptide described herein. Nucleic acid vectors consisting of such nucleic acid molecules are provided. Cells are provided that include the nucleic acid molecules or nucleic acid vectors described herein. In some embodiments, such cells express the BoNT polypeptide described herein.

Methods for producing a BoNT polypeptide according to the present disclosure are provided. Such methods include the steps of culturing a cell expressing BoNT polypeptides under conditions that produce said BoNT polypeptide. In some embodiments, the methods further comprise recovering the BoNT polypeptide from the culture.

In other aspects of the present disclosure, modified BoNT polypeptides are provided, consisting of (a) a protease domain; (b) a modified linker site and (c) a translocation domain; wherein (a), (b) and (c) belong to BoNT serotype X, and the modified linker region contains one single substitution type mutation at the position corresponding to C461 or C467 of SEQ ID NO: 1.

In some embodiments, the modified BoNT polypeptide further comprises (d) a receptor binding domain.

In some embodiments, the modified linker region contains a substitution type mutation corresponding to C461S or C461A in SEQ ID NO: 1. In certain embodiments, the modified linker region contains a mutation in

- 3 041568 replacement type corresponding to C467S or C467A in SEQ ID NO: 1.

In some embodiments, the receptor binding domain is a receptor from BoNT/X. In accordance with some embodiments, the receptor binding domain is modified. In some embodiments, the receptor binding domain contains a substitution mutation corresponding to C1240S or C1240A in SEQ ID NO: 1.

In some embodiments, the receptor binding domain is a receptor serotype selected from the group consisting of A, B, C, D, E, F, and G.

In some embodiments, the modified BoNT polypeptide enters the cell. In some embodiments, the modified BoNT polypeptide cleaves SNARE proteins in the cell. In some embodiments, the SNARE protein is selected from the group consisting of SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

In some embodiments, the SNARE protein is VAMP1. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 39.

In some embodiments, the SNARE protein is VAMP2. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 40.

In some embodiments, the SNARE protein is VAMP3. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO:41.

In some embodiments, the SNARE protein is VAMP4. In some embodiments, BoNT cleaves between amino acid residues corresponding to K87 and S88 of SEQ ID NO: 42.

In some embodiments, the SNARE protein is

VAMP5. According to some embodiments, BoNT cleaves between amino acid residues corresponding to R40 and S41 of SEQ ID NO:43.

In some embodiments, the SNARE protein is Ykt6. According to some embodiments, BoNT cleaves between amino acid residues corresponding to K173 and S174 of SEQ ID NO:44.

In some embodiments, the BoNT polypeptide has increased stability relative to its corresponding wild-type BoNT polypeptide.

In accordance with some embodiments, the cell is a secretory cell. In accordance with some embodiments, the cell is a neuronal cell. In some embodiments, the cell is an immune cell. In accordance with some embodiments, the BoNT polypeptide suppresses neuronal activity. In some embodiments, the BoNT polypeptide induces atrophic paralysis. In accordance with some embodiments, the cell is a cultured cell. In accordance with some embodiments, the cell is in vivo. In accordance with some embodiments, the cell is derived from a mammal. In some embodiments, the mammal is a human. In accordance with some embodiments, the mammal is a rodent. In accordance with some embodiments, the rodent is a mouse. In accordance with some embodiments, the rodent is a rat.

In some embodiments, the BoNT polypeptide does not cross-react with an antibody against BoNT serotype A, B, C, D, E, F, or G.

In accordance with some embodiments, the modified linker site contains an artificial linker. In accordance with some embodiments, the artificial linker contains a protease cleavage site. In some embodiments, the protease is selected from the group consisting of thrombin, TEV, PreScission (3C protease), factor Xa, MMP-12, MMP-13, MMP-17, MMP-20, granzyme-B, and enterokinase. In accordance with some embodiments, the implementation of the linker contains the amino acid sequence under any of SEQ ID NO: 50-60).

In other aspects of the present disclosure, nucleic acid molecules are provided, consisting of a polynucleotide encoding a polypeptide containing an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99%, or at least 99.5% or 100% identity with respect to the BoNT polypeptide described herein. Nucleic acid vectors consisting of such nucleic acid molecules are provided. Cells are provided that include the nucleic acid molecules or nucleic acid vectors described herein. In some embodiments, such cells express the BoNT polypeptide described herein.

Methods for producing a BoNT polypeptide according to the present disclosure are provided. Such methods include the steps of culturing a cell expressing BoNT polypeptides under conditions that produce said BoNT polypeptide. In some embodiments, the methods further comprise recovering the BoNT polypeptide from the culture.

In other aspects of the present disclosure, modified BoNT polypeptides are provided consisting of one or more substitution type mutations at positions corresponding to R360, Y363, H227, E228 or H231 in SEQ ID NO: 1. In accordance with some embodiments, one or more mutations at the replacement type corresponds to R360A/Y363F, H227Y, E228Q or H231Y in SEQ ID NO: 1.

In accordance with some embodiments, the modified BoNT polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 31-38. In some embodiments, the modified BoNT polypeptide contains an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to any sub

SEQ ID NO: 31-38, wherein the polypeptide does not have the amino acid sequence under SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the modified BoNT polypeptide consists of the amino acid sequence under any of SEQ ID NO: 31- 38.

In other aspects of the present disclosure, a modified BoNT/X polypeptide is provided, consisting of a) an inactive protease domain; b) a linker region; and c) a translocation domain. In accordance with some embodiments, the modified BoNT/X further comprises a receptor binding domain.

In some embodiments, the inactive protease domain contains one or more substitution mutations at positions corresponding to R360, Y363, H227, E228, or H231 of SEQ ID NO: 1. In some embodiments, one or more substitution mutations correspond to R360A/Y363F, H227Y, E228Q or H231Y of SEQ ID NO: 1.

In some embodiments, the modified BoNT polypeptide enters the cell. In some embodiments, the modified BoNT polypeptide does not cleave the SNARE protein.

In accordance with some embodiments, the modified BoNT/X polypeptide further comprises a modification in the linker region (b). In some embodiments, the modification in the linker region contains one single substitution type mutation at the position corresponding to C461 or C467 of SEQ ID NO: 1. In some embodiments, the single substitution type mutation corresponds to C461A, C461S, C467A, or C467S from SEQ ID NO: 1. In some embodiments, the modified BoNT/X polypeptide further comprises a modification in the receptor binding domain (d).

In some embodiments, the modification in the receptor binding domain comprises a substitution mutation at the position corresponding to C1240 of SEQ ID NO: 1.

In other aspects of the present disclosure, nucleic acid molecules are provided, consisting of a polynucleotide encoding a polypeptide containing an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99%, or at least 99.5 or 100% identity with respect to the BoNT polypeptide described herein. Nucleic acid vectors consisting of such nucleic acid molecules are provided. Cells are provided that include the nucleic acid molecules or nucleic acid vectors described herein. In some embodiments, such cells express the BoNT polypeptide described herein.

Methods for producing a BoNT polypeptide according to the present disclosure are provided. Such methods include the steps of culturing a cell expressing BoNT polypeptides under conditions that produce said BoNT polypeptide. In some embodiments, the methods further comprise extracting the BoNT polypeptide from

- 5 041568 culture.

In addition, the present document provides the use of the modified BoNT polypeptide described herein as a delivery vehicle for delivering therapeutic agents to neurons.

In some aspects of the present disclosure, chimeric molecules are provided consisting of a first moiety linked to a second moiety, wherein the first moiety is a modified BoNT polypeptide as described herein.

In accordance with some embodiments, the first part and the second part are covalently linked. In accordance with some embodiments, the first part and the second part are not covalently bonded.

In some embodiments, the second moiety is selected from the group consisting of a small molecule, a nucleic acid, a short polypeptide, and a protein. In accordance with some embodiments, the second part is a bioactive molecule. In some embodiments, the second portion is a drug other than the polypeptide. In some embodiments, the second moiety is a therapeutic polypeptide.

In other aspects of the present disclosure, nucleic acid molecules are provided, consisting of a polynucleotide encoding a polypeptide containing an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99%, or at least 99.5 or 100% identity to the chimeric BoNT polypeptide described herein. Nucleic acid vectors consisting of such nucleic acid molecules are provided. Cells are provided that include the nucleic acid molecules or nucleic acid vectors described herein. In some embodiments, such cells express a chimeric BoNT polypeptide as described herein.

Methods for producing a chimeric BoNT polypeptide according to the present disclosure are provided. Such methods include the steps of culturing a cell expressing the chimeric BoNT polypeptides under conditions that produce the chimeric BoNT polypeptide. In some embodiments, the methods further comprise recovering the chimeric BoNT polypeptide from the culture.

In other aspects of the present disclosure, pharmaceutical compositions are provided that consist of the BoNT polypeptides described herein.

In accordance with some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

Also provided is a kit consisting of such pharmaceutical compositions and directions for therapeutic administration of the pharmaceutical composition.

In some aspects of the present disclosure, methods for treating a condition are provided, comprising administering a therapeutically effective amount of a BoNT polypeptide, chimeric molecule, or pharmaceutical composition described herein to a subject for treating the condition.

In accordance with some embodiments, the condition is associated with overactive neurons or glands. In accordance with some embodiments, the condition is selected from the group consisting of spastic dysphonia, spastic torticollis, laryngeal muscle dystonia, oromandibular dystonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spastic colitis, neurogenic bladder, anismus, spasticity of the extremities, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other disorders of muscle tone, as well as other disorders characterized by involuntary movements of muscle groups, lachrymation, hyperhidrosis, increased salivation, excess gastrointestinal secretions, secretory disorders, pain due to muscle cramps, headache, dermatological or aesthetic/cosmetic conditions, and obesity/loss of appetite.

In accordance with some embodiments, the condition is not associated with unwanted neuronal activity. In accordance with some embodiments, the condition is selected from the group consisting of psoriasis, allergies, hemophagocytic lymphohistiocytosis, and alcoholic pancreatic diseases.

In accordance with some variants of implementation, the introduction is carried out by injection into the site where there is undesirable neuronal activity.

In the following aspects of the present disclosure, methods are provided for preparing a Clostridial botulinum neurotoxin (BoNT) polypeptide, the method comprising:

(i) obtaining a first BoNT fragment consisting of a light chain (LC) and an N-terminal heavy chain domain (H _N ), wherein the first BoNT fragment contains the C-terminal LPXTGG motif (SEQ ID NO: 60);

(ii) obtaining a second fragment of BoNT, consisting of the C-terminal domain of the heavy chain (H _C ); wherein the second BoNT fragment contains a specific protease cleavage site at its N-terminus;

(iii) cleavage of the second BoNT fragment with a specific protease, wherein the cleavage results in a free glycine residue at the N-terminus; and (iv) contacting the first BoNT fragment and the second BoNT fragment in the presence of a transpeptidase, thereby ligating the first BoNT fragment and the second BoNT fragment to form a ligated BoNT.

In accordance with some embodiments, the first BoNT fragment further comprises an affinity tag. In accordance with some embodiments, the affinity tag is fused to the first BoNT fragment at the N-terminus. In accordance with some embodiments, the affinity tag is fused to the first BoNT fragment at the C-terminus. In some embodiments, the affinity tag is selected from the group consisting of His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Softag 1, Softag 3, TC, V5, VSV, Xpress, Halo and Fc.

In accordance with some embodiments, the second BoNT fragment further comprises an affinity tag. In accordance with some embodiments, the affinity tag is fused to the first BoNT fragment at the N-terminus. In some embodiments, the affinity tag is fused to a second BoNT fragment at the C-terminus. In some embodiments, the affinity tag is selected from the group consisting of His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Softag 1, Softag 3, TC, V5, VSV, Xpress, Halo and Fc.

In some embodiments, the protease is selected from the group consisting of thrombin, TEV, PreScission, MMP-12, MMP-13, MMP-17, MMP-20, granzyme-B, enterokinase, and SUMO protease. In some embodiments, the cognate protease is thrombin.

In some embodiments, the first BoNT fragment is from BoNT serotype A, B, C, D, E, F, G, or X. According to some embodiments, the first BoNT fragment is from BoNT/X. In some embodiments, the second BoNT fragment is from BoNT serotype A, B, C, D, E, F, G, or X. According to some embodiments, the second BoNT fragment is from BoNT/A. In accordance with some embodiments, the second BoNT fragment is a BoNT/B fragment. In accordance with some embodiments, the second BoNT fragment is a BoNT/C fragment. In accordance with some embodiments, the second BoNT fragment is a BoNT/X fragment.

In some embodiments, the transpeptidase is a sortase. In some embodiments, the sortase is a Staphylococcus aureus (SrtA) sortase.

These and other aspects of the present disclosure, as well as various advantages and applications, will become apparent upon reference to the detailed disclosure of the present invention. As will become clear, each aspect of the present disclosure may cover various embodiments.

All documents referred to in this specification are incorporated herein by reference in their entirety.

Brief description of graphic materials

The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure that may be more readily understood by reference to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. A patent or application file contains at least one drawing in color. Copies of this patent publication or patent application with color graphic(s)(s) will be provided by the Office upon request and upon payment of the required fee.

In FIG. 1A-1E show the identification of BoNT/X as a new BoNT.

In FIG. 1A shows a phylogenetic tree of protein sequence alignments for BoNT/A-G, BoNT/F5, TeNT and BoNT/X analyzed by the ClustalW method. Percent sequence identity for each toxin and BoNT/X is indicated after each toxin. The percentages of sequence identity for BoNT/E and BoNT/F, as well as for BoNT/B and BoNT/G, were also noted.

In FIG. 1B, top panel, shows a schematic representation of three BoNT/X domains with a conserved protease motif in LC and a ganglioside binding motif in HC marked.

In FIG. 1B, lower panel, shows a sliding window of sequence comparison demonstrating that BoNT/X has a low degree of similarity evenly distributed over its

- 7 041568 sequence, in relation to all other seven BoNT and TeNT.

In FIG. 1C shows a schematic representation of an orf gene cluster that includes the BoNT/X gene (top panel) that has two unique features compared to the two known orfX cluster variants (middle and bottom panels): (1) there is an additional orfX2 protein (denoted as orfX2b) located next to the BoNT/X gene; (2) the reading frame of the orfX genes has the same direction as the BoNT/X gene.

In FIG. 1D schematically illustrates the unique gene targeting and additional OrfX2 gene found in BoNT/X.

In FIG. 1E shows the preliminary light chain structure of BoNT/X. The dark dot represents the active site of zinc. The structure is shown with a resolution of 1.9 A.

In FIG. 2A-2J show that LC from BoNT/X (X-LC) cleaves VAMP at a unique site.

In FIG. 2A shows X-LC, with and without EDTA pretreatment, incubated with extracts in rat brain surfactant (BDE). Western blot analysis was performed to detect syntaxin 1, SNAP-25 and VAMP2. Synaptophysin (Syp) was also detected as a loading control. LC from BoNT/A (A-LC) and BoNT/B (B-LC) were analyzed in parallel. Cleavage of VAMP2 with B-LC results in loss of immunoblot signals, while cleavage of SNAP-25 with A-LC generates a smaller fragment of SNAP-25 that can still be detected by immunoblot (marked with an asterisk). Incubation with X-LC results in loss of VAMP2 immunoblotting signals, indicating that X-LC has cleaved VAMP2. EDTA blocked the activity of X-, A- and B-LC.

In FIG. 2B shows VAMP2 (residues 1-96) purified as His6-tagged recombinant protein and incubated with X-LC. Samples were analyzed by SDS-PAGE and Coomassie blue staining. X-LC converted VAMP2 (1-96) into two smaller fragments, indicating that X-LC cleaved VAMP2.

In FIG. 2C-2E shows VAMP2 (1-96) incubated with X-LC. Whole protein samples were then analyzed by mass spectrometry (LC-MS/MS) to determine the exact molecular weight of the cleaved fragments. The peak values of the eluted peptide from the HPLC column were plotted in FIG. 2S per run time (RT, X axis).

Mass spectrometry data for two cleavage products are shown in FIG. 2D and 2E, respectively, with the mass-to-charge ratio (mass/charge) noted for each signal. The molecular weight was derived by multiplying m by z and then subtracting z. The protein sequences for the two cleavage products correspond to SEQ ID NOs: 61 and 62 from top to bottom and are shown in FIG. 2C.

In FIG. 2F shows the sequence alignment between VAMP 1, 2, 3, 4, 5, 7, 8, Sec22b and Ykt6, with the cleavage sites for BoNT/B, D, F, G and X underlined and the two SNARE motifs boxed. The sequences correspond to SEQ ID NO: 63-71 from top to bottom.

In FIG. 2G shows HA-labeled VAMP1, 3, 7 and 8 as well as myc-labeled Sec22b and Ykt6 expressed in HEK293 cells via transient transfection. Cell lysates were incubated with X-LC and subjected to immunoblot analysis, detecting the HA or Myc label. Actin served as a loading control. X-LC cleaved VAMP1, 3 and Ykt6 but not VAMP7, 8 and Sec22.

In FIG. 2H shows GST-labeled Ykt6 incubated with X-LC (100 nM) for the indicated time. Samples were analyzed by SDS-PAGE and Coomassie blue staining. X-LC cleaved Ykt6.

In FIG. 2I shows GST-labeled cytoplasmic domains from VAMP2 (33-86), VAMP4 (1-115) and VAMP5 (1-70) incubated with X-LC for the indicated time. Samples were analyzed by SDS-PAGE and Coomassie blue staining. X-LC cleaved both VAMP4 and VAMP5. A longer incubation time (360 min) is required to cleave the majority of VAMP5. Note that the VAMP5 protein contains an additional band, which is either a degradation product or a bacterial protein contaminant that is nearly equal (but not identical) to that of the SDS-PAGE cleavage product.

In FIG. 2J shows experiments carried out as described in FIG. 2A, except that VAMP4 and Sec22b were detected. Synaptotagmin I (Syt I) was detected as a loading control. X-LC split native VAMP4 into BDE.

In FIG. 3A-3E show the activation of BoNT/X by proteolytic cleavage of the linker site between LC and H _N .

In FIG. 3A shows the sequence alignment of the linker regions between LC and H _N of seven BoNTs and BoNT/X. The sequences corresponded to SEQ ID NO: 72-79 from top to bottom. BoNT/X has the longest linker region of any BoNT, which contains an additional cysteine in addition to the two conserved cysteines in LC and in H _N . The Lys-C restriction site in limited proteolysis was identified using a mass spectrometric approach.

In FIG. 3B shows cultured rat cortical neurons exposed to the indicated concentrations of X-LC-H _N in the medium for 12 hours. Cell lysates were collected and immunoblot analysis was performed to examine syntaxin 1, SNAP-25 and VAMP2 in neurons. Actin served as a

- 8 041568 loading control. Trypsin-activated LC-HNs from BoNT/A (A-LC-HN) and BoNT/B (B-LC-HN) were analyzed in parallel as controls. X-LC-HN entered neurons and cleaved VAMP2, as evidenced by the loss of VAMP2 immunoblotting signals. Lys-C activated X-LC-HN showed significantly higher efficacy than non-activated X-LC-HN. X-LC-HN is more efficient than trypsin-activated B-LC-HN and A-LC-HN, which did not show any detectable cleavage of their SNARE substrates in neurons at the same assay concentrations.

In FIG. 3C shows X-LC-HN mutants with the indicated mutated cysteine, as well as WT X-LC-HN activated by limited proteolysis and analyzed by SDS-PAGE and Coomassie blue staining, with or without DTT. The C423S mutation resulted in two 50 kDa fragments, with or without DTT. Mutants having C461S or C467S showed one band at 100 kDa in the absence of DTT, and it split into two ~50 kDa bands in the presence of DTT, demonstrating that both C461 and C467 in HN can form an interchain disulfide bond with C423 in LC. The X-LC-HN WT portion formed aggregates at the top of the SDS-PAGE gel. These aggregates are due to the formation of an intermolecular disulfide bond, since they disappeared in the presence of DTT. Mutation of any of the three cysteines destroys the aggregates, indicating that the formation of an intermolecular disulfide bond is due to the presence of an additional cysteine in the linker site. Most of the activated X-LC-HN WT also separated into two ~50 kDa bands on the SDS-PAGE gel without DTT. This is due to the disulfide bond rearrangement described in FIG. 3D.

In FIG. 3D shows X-LC-HN WT activated by limited proteolysis followed by re-incubation with indicated concentrations of NEM to block disulfide bond rearrangement. The samples were then analyzed by SDS-PAGE and Coomassie blue staining with or without DTT. The majority of WT X-LC-HN exists as a single band at 100 kDa without DTT after NEM treatment, indicating that WT X-LC-HN mainly contains an interchain disulfide bond.

In FIG. 3E shows experiments carried out as described in FIG. 3B, except that neurons were exposed to either WT or the indicated X-LC-HN mutants. Mutation of cysteine in LC (C423) abolishes X-LC-HN activity, while mutation of one of the two cysteines in H _N (C461 or C467) does not affect X-LC-H _N activity in neurons. These results confirm that interchain disulfide bond formation is essential for X-LC-HN activity.

In FIG. 4A-4F show that full length BoNT/X is active in cultured neurons and in vivo in mice. The sequences are: LVPR-GS (SEQ ID NO: 80), LPETGG-His6 (SEQ ID NO: 81), GG-His6 (SEQ ID NO: 82) and LPETGS (SEQ ID NO: 59).

In FIG. 4A is a schematic drawing illustrating the synthesis of full length BoNT/X using the sortase mediated ligation method.

In FIG. 4B shows that the sortase-mediated ligation reaction mixture and indicated control components were analyzed by SDS-PAGE and Coomassie blue staining. An asterisk indicates protein aggregates due to intermolecular disulfide bonds, since these aggregates disappeared in the presence of DTT. The molecular weight marker is in lane 1 (starting from the left side). Full length BoNT/X (X-FL) only appeared in the sortase-mediated ligation mix (lane 7 and lane 14).

In FIG. 4C shows that neurons were exposed to the same amount (5 μl) of the sortase-mediated ligation mixture or the indicated control components for 12 h in the medium. Cell lysates were analyzed by immunoblotting. X-LC-H _N separately cleaved some VAMP2 due to its high concentration in the reaction mixture. A control mixture containing both X-LC-H _N and X-HC but no sortase slightly enhanced VAMP2 cleavage compared to X-LC-H _N alone, probably because X-HC binds to X-LC- H _N through non-covalent interactions. Ligation of X-LC-H _N and X-HC with sortase enhanced VAMP2 cleavage in a mixture of X-LC-HN and X-HC without sortase, demonstrating that ligated X-FL is functional in neurons.

In FIG. 4D shows that the sortase reaction mixture prepared as described in panel b (lane 7) is active in vivo as analyzed using DAS analysis in mice. The injected limb developed atrophic paralysis, and the toes could not be extended for 12 hours. The left limb, which was not injected with toxins, served as a control.

In FIG. 4E shows that BoNT/AG, BoNT/DC mosaic toxin and BoNT/X were subjected to dot blot analysis (0.2 μg per toxin coated on nitrocellulose membranes) using four horse antisera (trivalent antibody against BoNT/A, B and E , antibodies against BoNT/C, antibodies against BoNT/DC and antibodies against BoNT/F), as well as two goat antisera (antibodies against BoNT/G and antibodies against BoNT/D). BoNT/X consists of purified X-LC-H _N and X-HC in a ratio of 1:1. These antisera recognized their respective target toxins, but none of them recognized BoNT/X.

In FIG. 4F shows that the inactive full length form of BoNT/X (BoNT/XRY) was purified as

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His6-tagged recombinant protein in E. coli and analyzed by SDS-PAGE and Coomassie blue staining, with or without DTT.

In FIG. 5 is a phylogenetic tree showing the distribution and relationship of clostridial neurotoxins. The tree represents the relationships of various BoNT and TeNT sequences from the JackHMMER search. BoNT/X is marked with a circle.

In FIG. 6 shows the mass spectrometry analysis of intact VAMP2 (1-96). His6-tagged VAMP2 (1-96) was analyzed by LC-MS/MS mass spectrometry. The HPLC profile is shown in the left panel along with the protein sequence. Full length mass spectrometry data for VAMP2 (1-96) are shown in the right panel and correspond to SEQ ID NO: 83 with the mass/charge value noted for each signal.

In FIG. 7A-7F show the identification of a cleavage site in GST-VAMP2 (33-86) by X-LC.

In FIG. 7A shows GST-tagged VAMP2 (33-86) incubated with or without X-LC. Samples were analyzed by SDS-PAGE and Coomassie blue staining.

In FIG. 7B, 7C show intact GST-labeled VAMP2 (33-86) analyzed by LC-MS/MS. The HPLC profile is shown in Fig. 7B. The mass spectrometry data is shown in FIG. 7C, with the protein sequence (SEQ ID NO: 84) marked in FIG. 7C. VAMP2 (33-66) and VAMP2 (67-86) are noted.

In FIG. 7D, 7E shows GST-tagged VAMP2 (33-86) incubated with X-LC. The samples were then analyzed by LC-MS/MS mass spectrometry. The HPLC profile is shown in Fig. 7D. Mass spectrometry data for the C-terminal fragment (SEQ ID NO: 85) generated by X-LC is shown in FIG. 7E.

Mass spectrometry data for the N-terminal fragment (SEQ ID NO: 86) are shown in FIG. 7F.

The protein sequences of the C- and N-terminal fragments are shown in FIG. 7E, 7F and correspond to SEQ ID NOs: 85 and 86, respectively.

In FIG. 8A, 8B show that the chimeric CA toxin is active in neurons.

In FIG. 8A shows a chimeric XA toxin formed by sortase-like ligation of X-LC-H _N to A-HC with X-FL as described in FIG. 4A. The sortase-mediated ligation mixture and indicated control components were analyzed by SDS-PAGE and Coomassie blue staining. The ligation is efficient because most of the X-LC-H _N has been ligated into the chimeric XA toxin.

In FIG. 8B shows rat cortical neurons exposed to the indicated controls or sortase-ligated CA mixture (5 µm) for 12 hours in medium. Cell lysates were analyzed by immunoblotting. X-LC-H _N separately cleaved some VAMP2 due to its high concentration in the reaction mixture. Ligated CA cleaved VAMP2 in neurons.

In FIG. 9 shows that mutating additional cysteine to H _N and cysteine to _HC does not affect BoNT/X activity. XH _C (C1240S) was ligated to WT X-LC-H _N , X-LC-H _N (C461S) or X-LC-H _N (C467S) by sortase-mediated ligation. Neurons were subjected to sortase-mediated ligation mixture or control components (5 μl) for 12 hours in medium. Cell lysates were analyzed by immunoblotting. Mutation of C1240 and one of the cysteines in H _N (C461 or C467) does not affect BoNT/X activity, since the ligated mutant toxins are able to enter neurons and cleave VAMP2.

In FIG. 10 shows an antiserum raised against seven BoNT serotypes neutralizing its target BoNTs in neurons. Cultured rat cortical neurons were subjected to the indicated BoNT, with or without re-incubation with the indicated antisera. Cell lysates were harvested 12 hours later and subjected to immunoblot analysis. All antisera specifically neutralized their target BoNTs without affecting the activity of the other BoNT serotype, thus confirming the specificity and efficacy of this antisera. BoNT concentrations were: BoNT/A (50 pM), BoNT/B (2 nM), BoNT/C (1.5 nM), BoNT/D (100 pM), BoNT/E (0.5 nM), BoNT/ F (0.5 nM), BoNT/G (5 nM). Antiserum against BoNT/A/B/E was used at 20 μl per well. The remaining antisera were used at 10 μl per well. BoNT was pre-incubated with the indicated antiserum for 30 min at 37°C before being added to the culture medium.

In FIG. 11A-11C show that BoNT/XRY is not active in neurons.

In FIG. 11A shows cultured rat cortical neurons exposed to BoNT/XRY at indicated concentrations. Cell lysates were analyzed by immunoblotting. VAMP2 was not cleaved, indicating that BoNT/XRY is not active in neurons.

In FIG. 11B shows SDS-PAGE analysis of BoNT/XRY expression in cell lysate and supernatant (S/N) (4-12% BisTris, MOPS buffer). The band at 150 kDa corresponding to BoNT/X is clearly visible both in the lysate and in the soluble fraction.

In FIG. 11C shows SDS-PAGE analysis of the final high purity BoNT/XRY sample (4-12% BisTris, MOPS buffer). One band at 150 kDa, corresponding to BoNT/X, is clearly visible and shows ~90% purity.

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In FIG. 12A-12F show that BoNT/X binds to all four brain gangliosides.

In FIG. 12A-12D show BoNT/X (squares) and A-HC (circles) binding to GD1a (FIG. 12A), GT1b (FIG. 12B), GD1b (FIG. 12C), and GM1 (FIG. 12D), respectively. Curves corresponded to the mean of triplicate ELISA analyzes and were plotted using Prism7 (GraphPad software).

In FIG. 12E is a summary of BoNT/X binding to all four gangliosides compared to complete BoNT/A binding in FIG. 12F.

In FIG. 13A-13D show the identification of X-LC cleavage sites in Ykt6 by mass spectrometry analysis.

In FIG. 13A-13D show that 10 μg of GST-labeled Ykt6 (1-192) with or without pre-incubation with X-LC was separated on SDS-PAGE (FIG. 13A). Protein strips were excised as indicated and digested with chymotrypsin. The digested peptides were desalted and analyzed by reverse phase HPLC through a C18 column combined with ESI-MS. HPLC profiles of GST-Ykt6 without pre-treatment with X-LC are shown in FIG. 13B, and a sample pretreated with X-LC is shown in FIG. 13C. It was found that the intensity of a single peptide was ~100 times higher in samples pre-treated with X-LC than in samples that were not subjected to X-LC (marked with an asterisk). This peptide was eluted after 37 min at room temperature with mass/charge = 611 (Fig. 13D), which could only correspond to the peptide sequence ESLLERGEKLDDLVSK (SEQ ID NO: 87) in Ykt6, indicating that this is a peptide located at N -terminal side of the cleavage site for X-LC. Therefore, the cleavage site is K173-S174 in Ykt6.

In FIG. 14A-14E show the identification of X-LC cleavage sites in VAMP4 and VAMP5 by mass spectrometry analysis. In FIG. 14A-14E show experiments performed as described in FIG. 13, except that VAMP4 (FIGS. 14B, 14C) and VAMP5 (FIGS. 14D, 14E) were analyzed.

In FIG. 14B shows a peptide that labels the N-terminal site of a cleavage site in VAMP4. The sequence of the DELQDK peptide corresponds to SEQ ID NO: 88.

In FIG. 14C shows a peptide that tags the C-terminal site of a cleavage site in VAMP4. The peptide sequence of SESLSDNATAF corresponds to SEQ ID NO: 89.

In FIG. 14D shows a peptide that labels the N-terminal site of a cleavage site in VAMP5. The sequence of the AELQQR peptide corresponds to SEQ ID NO: 90.

In FIG. 14E shows a peptide that tags the C-terminal site of a cleavage site in VAMP5. The peptide sequence SDQLLDMSSTF corresponds to SEQ ID NO: 91. Thus, cleavage sites were identified as K87-S88 in VAMP4 and R40-S41 in VAMP5.

Detailed disclosure of the present invention

Clostridium botulinum neurotoxins (BoNT) are a family of bacterial toxins produced by the bacterium Clostridium with seven well established serotypes (BoNT/AG) ^1-3 . They represent one of the most dangerous potential bioterrorist weapons classified by the United States Centers for Disease Control (CDC) as a Category A ⁴ Highly Threat. These toxins are produced as a single polypeptide and can be separated by bacterial or host proteases into a light chain (LC, ~50 kDa) and a heavy chain ( _HC , ~100 kDa). The two chains remain linked by an interchain disulfide bond. _HC contains two subdomains: an N-terminal _HN domain that mediates LC translocation across endosomal membranes, and a C-terminal He domain that mediates receptor binding in neurons. The interchain disulfide bond is repaired by LC translocation to the cytosol ^5, 6. The released LC acts as a protease with specific cleavage of a number of neuronal proteins: BoNT/A, C and E cleave at different sites in a protein known as SNAP-25; BoNT/B, D, F and G are cleaved at different sites in the VAMP vesicular protein; and BoNT/C also cleaves the transmembrane protein syntaxin 1 ¹ ' ³ . These three proteins form a complex known as the SNARE complex, which is required for the release of neurotransmitters ^7, 8. Cleavage of any of these three SNARE proteins blocks the release of neurotransmitters from neurons, thereby paralyzing muscles.

BoNTs are the most potent toxins known and cause diseases in humans and animals known as botulism ³ . The main form of botulism is caused by eating food contaminated with BoNT (food botulism). Other forms exist, such as infantile botulism, which is caused by colonization of the intestines by toxin-producing bacteria in children. BoNTs are always produced together with another 150 kDa protein known as NTNHA (non-toxic, non-hemagglutinin protein), which forms a pH dependent complex with BoNT and protects BoNT from proteases in the gastrointestinal tract ⁹ . Genes encoding BoNT and NTNHA have been found in two types of gene clusters: (1) HA cluster containing genes for three conserved proteins HA17, HA33, and HA70 that complex with BoNT/NTNHA and promote toxin absorption across the gut epithelial barrier ^10-12 ; (2) the OrfX cluster, which encodes conserved proteins OrfX1, OrfX2, OrfX3, and P47 with unknown functions ¹³ .

Because local injections of minute amounts of toxins can attenuate neuronal activity at targeted sites, BoNT has been used to treat a growing list of medical conditions, ^14-16 including muscle spasms, chronic pain, overactive bladder problems, and for cosmetic purposes. The BoNT market already exceeded $1.5 billion in 2011 and is projected to reach $2.9 billion by 2018.

BoNTs have traditionally been described using neutralization assays in mice by injecting a cultured supernatant of Clostridium bacteria into mice with or without antisera against known BoNTs. The first isolated serotypes, BoNT/A and BoNT/B, were established in 1919 by Georgina Burke ¹⁸ . The last of the seven types, BoNT/G, was identified in 1969 from soil samples in Argentina ¹⁹ . Since 1970, no new BoNT serotypes have been detected. This classification has been maintained since the protein sequences for each BoNT were determined in the 1990s. Sequence identity between any two pairs among the seven ranges from 32 to 65.3%. All seven BoNTs have been identified and characterized prior to the era of their medical applications. Therefore, there is no patent for any of these toxins. Any company is free to manufacture and sell any of these seven BoNTs. Among the seven types, BoNT/A and BoNT/B are the two toxins currently approved by the FDA for use in humans ^14,16 . BoNT/A is the dominant type used for both medical and cosmetic purposes, marketed as Botox by Allergan Inc., Dysport by IPSEN Inc. and Xeomin by Merz Inc. BoNT/B is sold as Myobloc by USWorld Med. There is considerable interest in the development of other types of BoNT as therapeutic toxins for two main reasons.

(1) The main limitation in treatment is the formation of a neutralizing antibody against BoNT/A or BoNT/B in patients, which makes future treatment with the same toxin ineffective ²⁰ . In this case, patients will need to be treated with a different type of BoNT. This is why BoNT/B is often used to treat patients who have developed neutralizing antibodies against BoNT/A during treatment, but there is a need for alternative toxins for patients who have developed antibodies against both BoNT/A and BoNT/B.

(2) Although all BoNTs have the same structure and function, there are also significant differences between them. For example, BoNT/A cleaves SNAP-25 and uses the SV2 protein as its receptor, while BoNT/B cleaves VAMP and uses the synaptotagmin (Syt) protein as its receptor ^21-27 . These functional variations may lead to potential differences in therapeutic efficacy targeting different types of neurons. In addition, the stability and duration of treatment may also differ among the seven types of toxins. Therefore, another type of toxin may have advantages over BoNT/A and BoNT/B.

Rapid advances in genome sequencing in recent years have revealed the amazing diversity of BoNT ^28,29 . First, there are several subtypes that can be recognized by the same antisera but contain significant levels of protein sequence variation (2.6-31.6% difference) ^{28, 30} For example, BoNT/A contains eight known subtypes, referred to as BoNT/A1-A8 ¹³ . In addition, there are many mosaic toxins, probably due to recombination of toxin genes. For example, type H was reported in 2013, but was later recognized as a chimeric toxin because its LC shares ~80% identity with LC subtype BoNT/F, BoNT/F5 and its _HC shares ~84% identity with _HC BoNT/ A1 ^31-34 . Accordingly, this toxin can be recognized and neutralized with available anti-BoNT/A ³³ antisera.

The gene cluster encoding BoNT may be located on plasmids, bacterial phages, or chromosomes, indicating that toxin genes are mobile and subject to horizontal gene transfer ¹³ . There are also cases where a strain of Clostridium bacteria contains two or even three different toxin genes ^{32, 35, 36} . In these cases, one toxin is usually expressed at higher levels (denoted by a capital letter) than the other toxin (denoted by a lowercase letter). For example, strains that express high levels of BoNT/B and low levels of BoNT/F are known as BoNT/Bf strains. There are also cases where one toxin is expressed and the other toxin is not expressed, which is known as a silent toxin (usually labeled in brackets). For example, a study of botulism cases in children in California found that 8% of the strains were BoNT/A(B), meaning that these strains contained genes for both BoNT/A and BoNT B, but only expressed at detectable levels BoNT/A ^37-39 .

As shown in the figures and examples of this disclosure, published databases were searched for Clostridium bacterial genomic sequences and a novel BoNT gene (hereinafter referred to as BoNT/X) encoded on the chromosome of Clostridium botulinum strain 111 was identified. Strain 111 was first isolated in Japan in a pediatric patient with botulism in 1996 ⁴⁰ . It was shown that the toxicity of strain 111 in mice can be neutralized by antiserum against BoNT/B ⁴⁰ . This strain was later confirmed to express the BoNT/B subtype, BoNT/B2, encoded in plasmid ^41,42 . The BoNT/X sequence was deposited in the PubMed database in February 2015 as part of the genomic sequence of strain 111. BoNT/X has not been previously characterized. It remains unknown whether it is expressed in strain 111 and whether it is a functional toxin.

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This document also presents the characterization of BoNT/X at functional levels. Its LC has been found to cleave VAMP at a site different from the known target sites of all other BoNTs. It was found that a full-length toxin, formed by covalent binding of non-toxic fragments by sortase, enters cultured neurons and cleaves VAMP in neurons, causing atrophic paralysis in mice. Finally, it was found that the toxin was not recognized by antisera raised against all seven known BoNTs, suggesting that BoNT/X is a new BoNT serotype. Its identification is an urgent task for the development of effective countermeasures. It can also be made into a new therapeutic toxin and can be used to create chimeric toxins with potentially different pharmacological properties.

As used herein, the term Clostridial botulinum neurotoxin (BoNT) polypeptide encompasses any polypeptide or fragment from the botulinum neurotoxin described herein. In accordance with some embodiments, the term BoNT refers to a full length BoNT. In some embodiments, the term BoNT refers to a fragment of BoNT that can carry out the entire cellular mechanism by which BoNT enters a neuron and inhibits the release of neurotransmitters. In some embodiments, the term BoNT simply refers to a BoNT fragment, which does not require the fragment to have any specific function or activity. For example, a BoNT polypeptide may refer to the light chain (LC) of BoNT, such as BoNT/X. Other terms that may be used throughout this disclosure for Clostridial botulinum neurotoxins may be BoNT, botulinum toxins, or C. botulium toxins. Please note that these terms are used interchangeably. The term BoNT/X refers to the new BoNT serotype described and characterized in the present disclosure. Also shown is the BoNT/X protein sequence (GenBank No. BAQ12790.1; the four cysteines are underlined and in bold):

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNTNDLNIPSEPIMEA DAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIPLPLVSNGALTLSDNETIAYQENNNIVSN LQANLVIYGPGPDIANNATYGLYSTPISNGEGTLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDP ASILMHELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYT TLISERLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKERPI DPIYWILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFIKICPRNGLLYNAIYRNSKNYLNN IDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYD FTEANSIPSISQQNILERNEELYEPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNP NKWSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNIGNDIRHGD FVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWWGTIHL QINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLS NSYLTKEMIPKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLI NQYKNEIEDYEVLNLGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATD nfsisfwikhpkptnllnngieytlvenfnq rgwkisiqdskliwylrdHNnsikivtpdyiafngwnlititn NRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLYNYYFNS NYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNS KKLDGLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIF HKSGLMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED (SEQ ID NO: I)·

The term modified Clostridial botulinum neurotoxin (BoNT) encompasses BoNT containing any modification in the amino acid sequence, such as truncation, addition, amino acid substitution, and any combination thereof. For example, BoNT/X containing amino acid substitution mutations in C461 or C467 is a modified BoNT. In another example, a full length BoNT fragment or domain (eg, protease domain or LC) is considered to be a modified BoNT. In some embodiments, the BoNT domain may also contain amino acid substitution mutations, such as a protease domain containing substitution mutations at positions C461 or C467 of BoNT/X.

The term enters the cell when used to describe the action of BoNT according to the present disclosure encompasses binding of BoNT to a low or high affinity receptor complex, binding of BoNT to a ganglioside, toxin internalization, translocation of the toxin light chain into the cytoplasm, and enzymatic modification of the BoNT substrate.

As used herein, the term Clostridial botulinum neurotoxin (BoNT) protease domain is synonymous with light chain (LC). The protease domain of BoNT is located in the light chain of BoNT and is thus also referred to as LC. The term refers to the BoNT domain, which

- 13 041568 which can carry out the stage of enzymatic target modification in the process of intoxication. When an LC from a specific BoNT serotype is mentioned, the term serotype-LC is used. For example, the term X-LC refers to the LC polypeptide from BoNT/X. The BoNT protease domain specifically targets a C. botulinum toxin substrate and proteolytically cleaves a C. botulinum toxin substrate, such as, for example, SNARE proteins such as SNAP-25 substrate, VAMP substrate, and syntaxin substrate, into BoNT (e.g., BoNT/X , BoNT/A BoNT/B, BoNT/C, etc.). The protease domain or LC is considered to correspond approximately to amino acids 1-439 in BoNT/X. The domain boundary can vary up to about 25 amino acids. For example, the protease domain may correspond to amino acids 1-414 or 1-464 in BoNT/X. In some embodiments, the protease domain may correspond to amino acids 1-438, 1-437, 1-436, 1-435, 1434, 1-433, 1-432, 1-431, 1-430, 1-429, 1 -439, 1-440, 1-441, 1-442, 1-443, 1-444, 1-445, 1-446, 1-447, 1-448 or 1-449 in BoNT/X.

As used herein, the term Clostridial botulinum neurotoxin translocation domain (BoNT) is synonymous with the term H _N domain and means a BoNT domain that can carry out the translocation step of an intoxication process that mediates BoNT light chain translocation. Thus, _HN facilitates the movement of the BoNT light chain across the membrane into the cell cytoplasm. Non-limiting examples of H _N include H _N BoNT/A, H _N BoNT/B, H _N BoNT/Cl, H _N BoNT/D, H _N BoNT/E, H _N BoNT/F, H _N BoNT/G, and H _NBoNT /X. The translocation domain is located at the N-terminus of the heavy chain (HC) and is thus also referred to as H _N . Please note that these terms are used interchangeably in this document.

As used herein, the term linker site refers to the amino acid sequence between the protease domain and the BoNT translocation domain. The linker contains two cysteines at positions 461 and 467, one of which forms an intermolecular disulfide bond with a cysteine in the C423 protease domain (C461-C423 disulfide bond or C467C423 disulfide bond). The formation of this disulfide bond is essential for BoNT/X activity.

As used herein, the term LC-H _N refers to a BoNT polypeptide containing a protease domain, a linker region and a translocation domain. If LC-H _N from a particular BoNT serotype is mentioned, then the term serotype-LC-HN is used. For example, the term X-LC-H _N means the LC-H _N polypeptide from BoNT/X. The LC-H _N polypeptide is believed to correspond approximately to amino acids 1-892 from BoNT/X. The domain boundary can vary up to about 25 amino acids. For example, an LC-H _N polypeptide may correspond approximately to amino acids 1-917 or 1-867 in BoNT/X. In some embodiments, the LC-H _N polypeptide may correspond to amino acids 1-893, 1-894, 1-895, 1-896, 1-897, 1-898, 1-899, 1-900, 1-901, 1-902, 1-892, 1-891, 1-890, 1-889, 1-888, 1-887, 1-886, 1-885, 1-884 or 1-883 in BoNT/X.

As used herein, the term Clostridial botulinum neurotoxin (BoNT) receptor-binding domain is synonymous with the term _HC domain and refers to any naturally occurring BoNT receptor-binding domain that can perform a cell-binding step during intoxication, including, for example, binding BoNT with a BoNT-specific receptor system located on the surface of the plasma membrane of the target cell. Some aspects of the present disclosure relate to modified BoNT receptor binding domains from serotype X (BoNT/X). In some embodiments, the modified BoNT/X receptor binding domain contains amino acid substitutions at the position corresponding to C1240 in BoNT/X (SEQ ID NO: 1). The receptor or HC binding domain is considered to correspond approximately to amino acids 893-1306 of BoNT/X. The domain boundary can vary up to about 25 amino acids. For example, the receptor or HC binding domain may correspond to amino acids 868-1306 or 918-1306. In some embodiments, the receptor or HC binding domain may correspond to amino acids 893-1306, 894-1306, 895-1306, 896-1306, 897-1306, 898-1306, 899-1306, 900-1306, 901-1306 , 902-1306, 892-1306, 891-1306, 890-1306, 889-1306, 888-1306, 887-1306, 886-1306, 885-1306, 884-1306 or 883-1306 in BoNT/X.

The term isolated means material that does not contain, to varying degrees, the components that normally accompany it in its native state. The term isolate means the degree of isolation from the original source or environment, such as from a cell, or from flanking DNA, or from a natural source of DNA. The term "purified" is used to refer to a substance, such as a polypeptide, that is substantially pure in relation to other components of the formulation (eg, other polypeptides). It may refer to a polypeptide that is at least about 50%, 60%, 70%, or 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure in in relation to other components. The terms substantially pure or substantially pure, in relation to a polypeptide, refers to a formulation that contains less than about 20%, more preferably less than about 15, 10, 8, 7%, most preferably less than about 5, 4, 3, 2 , 1 or less than 1% of one or more others

- 14 041568 components (eg other polypeptides or cellular components).

The term substitution mutation, without reference to a specific amino acid, can include any amino acid other than the wild-type residue normally found at that position. Such substitutions may be replaced by non-polar (hydrophobic) amino acids such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Substitutions can be replaced by polar (hydrophilic) amino acids such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The substitutions can be replaced by electrically charged amino acids, for example negatively electrically charged amino acids such as aspartic acid and glutamic acid, and positively electrically charged amino acids such as lysine, arginine and histidine.

The substitution mutations described herein will generally be replaced by another naturally occurring amino acid residue, but in some cases non-naturally occurring amino acid residues may also be substituted. Non-natural amino acids, as used herein, are non-proteinogenic (ie, non-coding for protein) amino acids that are either naturally occurring or chemically synthesized. Examples include, without limitation, β-amino acids (β3 and β2), homoamino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear amino acids, diamino acids, D-amino acids and N-methylamino acids. In accordance with some embodiments, the amino acid may be substituted or unsubstituted. The substituted amino acid or substituent may be a halogenated aromatic or aliphatic amino acid, a halogenated aliphatic or aromatic modification in a hydrophobic side chain, or an aliphatic or aromatic modification.

The term percent identity between two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. sci. USA, 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. sci. USA, 90:5873-77, 1993. Such an algorithm is included in the NBLAST and XBLAST programs (version 2.0) Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of interest. If there are gaps between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

Therefore, in some aspects of the present disclosure, isolated BoNT polypeptides are provided. In some embodiments, the isolated BoNT polypeptide is a full length BoNT/X polypeptide. In some embodiments, the isolated BoNT polypeptide contains the amino acid sequence under SEQ ID NO: 1. In some embodiments, the isolated BoNT/X polypeptide contains an amino acid sequence that shares at least 85% identity with SEQ ID NO: 1. For example, an isolated BoNT polypeptide may contain an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least at least 99% or at least 99.5% identity to SEQ ID NO: 1. In some embodiments, the isolated BoNT polypeptide contains an amino acid sequence that is characterized by 8 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 100% identical to SEQ ID NO: 1. According to some in embodiments, the isolated BoNT polypeptide consists of the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the isolated BoNT polypeptide is an X-LC-H _N polypeptide. In some embodiments, the isolated BoNT polypeptide contains the amino acid sequence of SEQ ID NO: 2. In some embodiments, the isolated BoNT polypeptide contains an amino acid sequence that shares at least 85% identity with SEQ ID NO: 2. For example, , an isolated BoNT polypeptide may contain an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to SEQ ID NO: 2. In some embodiments, the isolated BoNT polypeptide contains an amino acid sequence that characterizes 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 100% identical to SEQ ID NO: 2. According to In some embodiments, the isolated BoNT polypeptide consists of the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the isolated BoNT polypeptide is an X-LC polypeptide. In some embodiments, the isolated BoNT polypeptide contains the amino acid sequence under SEQ ID NO: 3. In some embodiments, the isolated BoNT polypeptide contains an amino acid sequence that shares at least 85% identity with SEQ ID NO: 3. For example, , an isolated BoNT polypeptide may contain an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to SEQ ID NO: 3. In some embodiments, the isolated BoNT polypeptide contains an amino acid sequence that is characterized by 85.8 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 100% identical to SEQ ID NO: 3. In accordance with some embodiments the isolated BoNT polypeptide consists of the amino acid sequence of SEQ ID NO: 3.

An X-LC polypeptide can be introduced alone into cells where cleavage of a BoNT substrate (e.g., SNARE protein) is desired for research or therapeutic purposes, by any of the exogenous protein expression techniques known in the art, for example, transfection of an LC-coding sequence directly into cells, using lentiviral vectors, using AAV vectors, or X-LC fusion with cell-penetrating peptides.

In some embodiments, the BoNT polypeptides of the present disclosure are full length BoNT/X containing a protease domain (LC), a linker region, a translocation domain (H _N ), and a receptor binding domain (H _C ), wherein the linker the site is located between the protease domain and the translocation domain. Like other BoNTs, BoNT/X is initially produced as a single polypeptide and is activated by cleavage of the linker site between LC and H _N by either bacterial proteases or host proteases. This process is known as activation and is required for BoNT/X activity after cleavage, with the LC and H _N remaining connected by an interstrand disulfide bond until the LC translocation into the cytosol of the cells, where the disulfide bond is restored to release the LC into the cytosol. BoNT/X contains two cysteines that are conserved compared to other BoNTs, C423 and C467. Interestingly, BoNT/X also contains an additional cysteine (C461) that is unique to BoNT/X. The formation of an interchain disulfide bond (C423-C461 or C423-C467) is required for BoNT/X activity.

In addition to the cysteines in the linker region, the BoNT receptor binding domain contains another cysteine, C1240, which can also form intermolecular disulfide bonds with other cysteines in BoNT/X. These intermolecular disulfide bonds cause BoNT/X to aggregate and destabilize the protein (FIG. 4B). Substitution of cysteines that are not essential for BoNT/X activity can produce BoNT/X polypeptides with increased stability.

Therefore, in some aspects of the present disclosure, a modified BoNT/X polypeptide containing one or more substitution type mutations in C461, C467, or C1240 is provided that is more stable than wild-type BoNT/X and has comparable activities. The cysteines can be replaced by any amino acid that abolishes the formation of disulfide bonds. In some embodiments, the cysteines are replaced with serine (S) or alanine (A). Possible combinations of substitution mutations that may be present in modified BoNTs according to the present disclosure are, but are not limited to, C461S, C461A, C467S, C467A, C1240S, C1240A, C461S/C1240S, C461A/C1240S, C461S/C1240A,

C467A/C1240A, C467S/C1240S, C467A/C1240S, C467S/C1240A and C467A/C1240A. The / sign indicates double mutations. In some embodiments, the modified BoNT/X polypeptide of the present disclosure comprises the amino acid sequence of any one of SEQ ID NOs: 4-17. In some embodiments, the modified BoNT/X polypeptide contains an amino acid sequence that shares at least 85% identity with any of SEQ ID NO: 4-17 and does not have an amino acid sequence of SEQ ID NO: 1. For example, a modified BoNT/X polypeptide may contain an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least at least 99% or at least 99.5% identity with respect to any under SEQ ID NO: 4-17, and does not have the amino acid sequence under SEQ ID NO: 1.

- 16 041568 according to some embodiments, the modified BoNT/X polypeptide contains an amino acid sequence that is characterized by 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99, 99.5, or 100% identity to any under SEQ ID NO: 4-17, and does not have the amino acid sequence under SEQ ID NO: 1. In some embodiments, the modified polypeptide BoNT/X consists of the amino acid sequence of any one of SEQ ID NOs:4-17.

In some embodiments, the modified BoNT polypeptide of the present disclosure is a modified BoNT/X-LC-H _N polypeptide containing the substitution mutations described herein. In some embodiments, the modified BoNT/X-LC-H _N contains one single substitution type mutation at the position corresponding to C461 or C467 in SEQ ID NO: 2. In some embodiments, the modified BoNT/X-LC-H _N contains one single substitution mutation corresponding to C461A, C461S, C467A, or C467S in SEQ ID NO: 2. In some embodiments, the modified BoNT/X polypeptide of the present disclosure comprises the amino acid sequence of any of SEQ ID NO: 18-21. In some embodiments, the modified BoNT/X-LC-H _N polypeptide contains an amino acid sequence that shares at least 85% identity with any of SEQ ID NOs: 18-21 and does not have an amino acid sequence of SEQ ID NOs: 18-21. NO: 2. For example, a modified BoNT/X-LC-H _N polypeptide may contain an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99%, or at least 99.5% identity with respect to any under SEQ ID NO: 18-21, and does not have the amino acid sequence under SEQ ID NO: 2. In accordance with in some embodiments, the modified BoNT/X-LC-H _N polypeptide contains an amine an acid sequence that is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 100% identical to any of SEQ ID NO : 18-21, and does not have the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified BoNT/X-LC-H _N polypeptide consists of the amino acid sequence of any of SEQ ID NO: 18-21.

A modified BoNT polypeptide containing one or more substitution type mutations (e.g., in C461, C467, or C1240) described herein does not form intermolecular disulfide bonds that cause protein aggregation and is therefore more stable than its corresponding wild-type proteins. type. The activity of BoNT polypeptides is not disturbed by substitution mutations in cysteines. Thus, modified BoNT/X may be more suitable for therapeutic use than wild-type BoNT/X due to its increased stability.

In other aspects of the present disclosure, chimeric BoNTs are provided comprising the BoNT/X-LC-H _N described herein and a receptor binding (HC) domain from another BoNT. For example, the receptor binding domain can be any of BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/E, BoNT/F, and BoNT/G. Thus, the chimeric BoNTs described herein include BoNT/X-LC-H _N -AH _C , BoNT/X-LC-H _N -BH _C , BoNT/X-LC-H _n -CH _c , BoNT/X-LC-H _n -DH _c , BoNT/X-LC-H _n -EH _c , BoNT/X-LC-H _n -FH _c , and BoNT/X-LC-H _N -GH _C . It should be appreciated that the H _C domain of any subtypes of the seven known serotypes (eg, A, B, C, D, E, F or G) is suitable for the chimeric toxin. Reference to BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F or BoNT/G covers all subtypes. For example, BoNT/A has eight subtypes, BoNT/A1, BoNT/A2, BoNT/3, BoNT/A4, BoNT/A5, BoNT/A6, BoNT/A7 or BoNT8, and H _C of any of these subtypes

BoNT/A is suitable for use in a chimeric BoNT according to the present disclosure. Similarly, H _C of any of the eight BoNT/B subtypes, ie. BoNT/B1, BoNT/B2, BoNT/B3, BoNT/B4, BoNT/B5, BoNT/B6, BoNT/B7 or BoNT/B8 is suitable for use in chimeric BoNT according to the present disclosure.

According to some embodiments, BoNT/X-LC-H _N -A1-H _C (SEQ ID NO: 22), BoNT/X-LC-H _N- B1-H _C (SEQ ID NO: 23) and BoNT /X-LC-H _n -C1-H _c (SEQ ID NO: 24). In some embodiments, the chimeric BoNT polypeptide contains an amino acid sequence that shares at least 85% identity with any of SEQ ID NOs: 22-24. For example, a chimeric BoNT polypeptide may comprise an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % or at least 99.5% identity with respect to

- 17 041568 to any under SEQ ID NO: 22-24. In some embodiments, the chimeric BoNT polypeptide contains an amino acid sequence that is characterized by 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 100% identical to any sub

SEQ ID NO: 22-24. In some embodiments, the chimeric polypeptide

BoNT consists of the amino acid sequence of any one of SEQ ID NOs: 22-24.

In some embodiments, a chimeric BoNT according to the present disclosure includes a modified BoNT/X-LC-H _N containing a substitution mutation at the linker site, for example, at the position corresponding to C461 or C467 of SEQ ID NO: 2 For example, BoNT/X-LC-H _N in a chimeric BoNT may contain a substitution mutation corresponding to C461A, C467A, C461S, or C467S in SEQ ID NO: 2. For example, a chimeric BoNT polypeptide of the present disclosure may contain the amino acid sequence under any of SEQ ID NO: 25-30. In some embodiments, the chimeric BoNT polypeptide comprises an amino acid sequence that shares at least 85% identity with any of SEQ ID NOs: 25-30. For example, a chimeric BoNT polypeptide may comprise an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % or at least 99.5% identity with respect to any under SEQ ID NO: 25-30. In some embodiments, the chimeric BoNT polypeptide contains an amino acid sequence that is characterized by 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 100 % identity with respect to any under SEQ ID NO: 25-30. In accordance with some embodiments, the chimeric BoNT polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 25-30.

To create chimeric toxins, for example the BoNT/X-LC-H _N -A1-H _C toxin, an X-LC-H _N fragment containing amino acid approximately 1-892 (SEQ ID NO: 2) is fused to the receptor binding domain of any from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/E, BoNT/F and BoNT/G. The receptor binding domains of other BoNTs corresponded to amino acids approximately 860-1291 in BoNT/B1. It should be taken into account that the boundary of the X-LC-H _N fragment and/or receptor binding domains may vary by 1-25 amino acids. For example, an X-LC-H _N fragment that can be used for a chimeric toxin may contain amino acids 1-917 or 1-867 of BoNT/X. In some embodiments, an X-LC-HN fragment that can be used for a chimeric toxin may contain amino acids 1-893, 1-894, 1-895, 1-896, 1-897, 1-898, 1- 899, 1-900, 1901, 1-902, 1-892, 1-891, 1-890, 1-889, 1-888, 1-887, 1-886, 1-885, 1-884 or 1- 883BoNT/X. Similarly, a receptor binding domain that can be used for a chimeric toxin may contain an amino acid corresponding to 885-1291 or 835-1291 BoNT/X. In some embodiments, the receptor binding domain that can be used for the chimeric toxin may comprise an amino acid corresponding to 860-1291, 861-1291, 862-1291, 863-1291, 864-1291, 865-1291, 1291 867-1291 868-1291 869-1291 870-1291 860-1291 859-1291 858-1291 857-1291 856-1291 855-1291 852-1291 or 851-1291 BoNT/B. One skilled in the art will be able to identify domains that can be used for a chimeric toxin according to the present disclosure, based on their knowledge of protein homology, with or without sequence alignment software. Fusion fusion techniques are standard recombinant techniques that are well known to those skilled in the art.

The following describes modified BoNT/X polypeptides containing a modified linker site, wherein the linker site contains a specific protease cleavage site. As used herein, the term specific protease cleavage site refers to a recognition and cleavage site for a specific protease, as opposed to a sequence that is recognized and cleaved by more than one non-specific protease. Such specific proteases include, without limitation, thrombin, TEV, PreScission, factor Xa, MMP-12, MMP-13, MMP-17, MMP-20, granzyme-B, and enterokinase. A specific protease cleavage site can be added to the linker region of the BoNT/X polypeptide by inserting or substituting existing amino acids in the linker region (eg, substitutions of amino acids 424-460 in the BoNT/X polypeptide). The sequences of specific protease cleavage sites are also shown: LVPR|GS (thrombin, SEQ ID NO: 50), ENLYFQ|G (TEV, SEQ ID NO: 51), LEVLFQ|GP (PreScission, SEQ ID NO: 52), IEGR| or IDGR| (factor Xa, SEQ ID NO: 53 or 54), DDDDK| (enterokinase, SEQ ID NO: 55) and AHREQIGG| (SUMO protease, SEQ ID NO: 56). Sign | indicates where splitting occurs.

In other aspects of the present disclosure, functional characterization of BoNT/X polypeptides is provided. BoNT/X polypeptides, modified BoNT/X polypeptides, and chimeric BoNT polypeptides of the present disclosure can bind to and enter target cells, such as neurons, and cleave their substrate proteins, such as SNARE proteins. As used herein, the term SNARE proteins refers to SNAP (soluble NSF-associated protein) receptors, which are a large superfamily of more than 60 proteins in yeast and mammalian cells. The main role of SNARE proteins is to mediate vesicle fusion, i.e. fusion of vesicles with their target membrane-bound compartments (such as the lysosome). The most studied SNARE proteins are those that mediate docking of synaptic vesicles to the presynaptic membrane in neurons, such as SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, VAMP7, VAMP8, syntaxin 1, and Ykt6. Some of these SNARE proteins are BoNT substrates. For example, VAMP1, VAMP2, VAMP3, SNAP-25 and syntaxin 1 have been shown to be cleaved by known BoNTs such as BoNT/A and BoNT/B.

This document presents data demonstrating that BoNT/X cleaves SNARE proteins, which are known substrates of BoNT. One surprising discovery of the present invention is that BoNT/X is able to cleave some SNARE proteins that other BoNTs cannot cleave, such as VAMP4, VAMP5 and Ykt6. VAMP4 is widely expressed and is known to mediate vesicular fusion between the trans-Golgi network (TGN) and endosomes, as well as homotypic fusion of endosomes. Traditionally, BoNTs are known to be limited to targeted SNAREs that mediate vesicle exocytosis on plasma membranes. BoNT/X is the first BoNT capable of cleaving SNARE, which mediates other types of fusion events within cells that by definition lack a plasma membrane. VAMP4 may also contribute to asynchronous synaptic vesicle exocytosis, growthosomal exocytosis, and activity-dependent volumetric endocytosis (ADBE) in neurons. In addition, VAMP4 is involved in the release of granules in immune cells. Thus, BoNT/X may have a unique potential among all BoNTs to modulate inflammatory secretion in immune cells, which can be used therapeutically. VAMP5 is predominantly expressed in muscle and its function has not yet been established. BoNT/X will be a unique tool to investigate the function of VAMP4 and VAMP5. Ykt6 functions in the endoplasmic reticulum for Golgi transport. It also functions in the early/recycling endosome in TGN transport. The identification of Ykt6 as a substrate for the BoNT polypeptides described herein is important as it opens up a new therapeutic application for blocking secretion in a wide range of cells with BoNT.

Another surprising finding of the present invention is that BoNT/X cleaves SNARE proteins at a new site that has not been previously described. As shown in the examples and graphics of the present invention, BoNT/X cleaves between the amino acids R66-S67 in VAMP1, VAMP2 and VAMP3. R66-A67 is a new cleavage site, different from the established target sites for all other BoNTs (FIG. 2F). It is also the only BoNT cleavage site located in the region previously known as the SNARE motif (Fig. 2F).

Therefore, the BoNT polypeptides of the present disclosure have an enhanced profile of target cells and substrates. In some embodiments, the BoNT polypeptide cleaves the SNARE protein in the cell. In some embodiments, the BoNT polypeptide cleaves a SNARE protein selected from the group consisting of SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1. In some embodiments, the BoNT polypeptide cleaves VAMP1 (SEQ ID NO: 39). In some embodiments, the BoNT polypeptide cleaves VAMP1 between the amino acid residues corresponding to R66 and A67 in SEQ ID NO: 39. In some embodiments, the BoNT polypeptide cleaves VAMP2 (SEQ ID NO: 40). In some embodiments, the BoNT polypeptide cleaves VAMP2 between the amino acid residues corresponding to R66 and A67 in SEQ ID NO: 40. In some embodiments, the BoNT polypeptide cleaves VAMP3 (SEQ ID NO: 31). In some embodiments, the BoNT polypeptide cleaves VAMP3 between the amino acid residues corresponding to R66 and A67 in SEQ ID NO: 41. In some embodiments, the BoNT polypeptide cleaves VAMP4 (SEQ ID NO: 42). In some embodiments, the BoNT polypeptide cleaves VAMP4 between the amino acid residues corresponding to K87 and S88 in SEQ ID NO: 42. In some embodiments, the BoNT polypeptide cleaves VAMP5 (SEQ ID NO: 43). In some embodiments, the BoNT polypeptide cleaves VAMP5 between the amino acid residues corresponding to R40 and S41 in SEQ ID NO: 43. In some embodiments, the BoNT polypeptide cleaves Ykt6 (SEQ ID NO: 44). In some embodiments, the BoNT polypeptide cleaves Ykt6 between the amino acid residues corresponding to K173 and S174 in SEQ ID NO: 44.

In some embodiments, the BoNT polypeptide of the present disclosure cleaves a SNARE protein in the target cell. As used herein, the term target cell means a cell, which is a naturally occurring cell into which BoNT can enter or which can intoxicate. In some embodiments, the target cell is a secretory cell, such as a neuron or a secretor

- 19 041568 new immune cell. Examples of neurons that can be BoNT target cells include, without limitation, motor neurons; sensory neurons; autonomous neurons; such as, for example, sympathetic neurons and parasympathetic neurons; non-peptidergic neurons, such as, for example, cholinergic neurons, adrenergic neurons, noradrenergic neurons, serotonergic neurons, GABAergic neurons, and peptidergic neurons, such as, for example, substance P neurons, calcitonin-related peptide neurons, gut vasoactive peptide neurons, neuropeptide neurons Y, cholecystokinin neurons.

A BoNT polypeptide of the present disclosure, such as BoNT/X or a modified BoNT/X polypeptide, is able to target secretory cell types other than neurons due to its ability to cleave VAMP4 or Ykt6. In some embodiments, the secretory cell targeted by the BoNT polypeptide is a secretory immune cell. As used herein, the term secretory immune cell refers to immune cells that secrete cytokines, chemokines, or antibodies. Such secretory immune cells can be innate immune cells, including, but not limited to, natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. Secretory immune cells that secrete antibodies (eg, white blood cells) can also be targeted with BoNT polypeptides according to the present disclosure. Non-limiting examples of antibody-secreting cells include, without limitation, plasma B cells, plasma cells, plasma cells, and effector B cells. In some embodiments, the target cell is a cultured cell, such as a cultured neuron or a cultured secretory immune cell. In accordance with some embodiments, the target cell is in vivo. In accordance with some embodiments, the target cell is a mammalian cell. In some embodiments, the mammal is a human. According to embodiments, the mammal is a rodent, such as a mouse or a rat.

In accordance with some embodiments, the BoNT polypeptide suppresses neuronal activity. In some embodiments, the BoNT polypeptide modulates an immune response. According to some embodiments, the BoNT polypeptide induces atrophic paralysis. The term atrophic palsy refers to a clinical manifestation characterized by weakness or paralysis and reduced muscle tone without another apparent cause (eg trauma).

In other aspects of the present disclosure, modified BoNT/X polypeptides containing an inactive protease domain are provided. Such BoNT/X polypeptides (also referred to herein as inactive BoNT/X) can enter target cells but cannot cleave substrate proteins (eg SNARE protein) due to inactive protease domain. According to some embodiments, the inactive BoNT/X is an X-LC-H _N fragment containing a) an inactive protease domain; b) a linker site; and c) a translocation domain. In some embodiments, the inactive BoNT/X is a full length BoNT/X polypeptide comprising a) an inactive protease domain; b) linker site; c) a translocation domain; and d) a receptor binding domain. In some embodiments, the inactive protease domain contains one or more substitution type mutations at position corresponding to R360, Y363, H227, E228, or H231 in SEQ ID NO: 1. In some embodiments, one or more mutations at type of replacement correspond to R360A/Y363F,

H227Y, E228Q or H231Y in SEQ ID NO: 1. Note that an inactive BoNT/X polypeptide may contain any mutation(s) that inactivates the protease domain.

In some embodiments, the inactive BoNT/X polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 31-38. In some embodiments, the inactive BoNT/X polypeptide contains an amino acid sequence that shares at least 85% identity with any sub

SEQ ID NOs: 31-38. For example, an inactive BoNT/X polypeptide may contain an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with respect to any sub

SEQ ID NO: 31-38. In some embodiments, the inactive polypeptide

BoNT/X contains an amino acid sequence that is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 100% identical to any under

SEQ ID NOs: 31-38. According to some embodiments, the inactive BoNT/X polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 31-38.

In accordance with some embodiments, an inactive BoNT/X (e.g., an inactive

- 20 041568 ny X-LC-HN or inactive BoNT/X full length) additionally contains mutations in the linker region. In some embodiments, the modification in the linker region contains one single substitution type mutation at the position corresponding to C461 or C467 of SEQ ID NO: 1. In some embodiments, the single substitution type mutation corresponds to C461A, C461S, C467A, or C467S in SEQ ID NO: 1. In some embodiments, the inactive BoNT/X (eg, inactive full length BoNT/X) further comprises a modification in the receptor binding domain. In some embodiments, the modification in the receptor binding domain comprises a substitution mutation at the position corresponding to C1240 in SEQ ID NO: 1.

It is also contemplated that a modified BoNT/X polypeptide containing an inactive protease domain described herein can be used as a delivery tool to target cells (eg, neurons) in humans. For example, the modified BoNT/X can be linked to other therapeutics, covalently or non-covalently, and acts as a targeting vehicle to deliver the therapeutics to target cells in humans.

Thus, another aspect of the present disclosure relates to a chimeric polypeptide molecule containing a first moiety, which is an inactive BoNT/X containing one or more substitution type mutations that inactivate the protease domain associated with the second moiety. The second moiety may be a bioactive molecule, such as a therapeutic agent (eg, a polypeptide or non-polypeptide drug). The binding of the first and second moieties may be covalent (eg, in the form of a fusion protein) or non-covalent. Methods for such binding are known in the art and can be easily applied by a person skilled in the art. If the second portion of the chimeric molecule is a polypeptide and the chimeric molecule is in the form of a protein, then nucleic acids and nucleic acid vectors encoding such chimeric molecules are provided.

Cells containing nucleic acids or nucleic acid vectors and cells expressing such chimeric molecules are also provided. Chimeric molecules in the form of a fusion protein can be expressed and isolated using the methods disclosed herein.

Modified BoNT/X polypeptides, chimeric BoNT polypeptides, or chimeric molecules containing a second moiety that is a polypeptide according to the present disclosure (e.g., without limitation, polypeptides containing the amino acid sequence of any of SEQ ID NOs: 1-38), as typically will be produced by expression of recombinant nucleic acids in appropriate cells (eg E. coli or insect cells) and isolated. Nucleic acids encoding the polypeptides described herein can be obtained and the nucleotide sequence of the nucleic acids can be determined by any method known in the art.

Also provided herein are isolated and/or recombinant nucleic acids encoding any of the BoNT polypeptides disclosed herein. Nucleic acids encoding isolated polypeptide fragments according to the present disclosure may be DNA or RNA, double stranded or single stranded. In some aspects, contemplated nucleic acids encoding isolated polypeptide fragments, as will be further understood, include nucleic acids encoding polypeptides that are variants of any of the modified BoNT polypeptides described herein.

Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants. In some embodiments, an isolated nucleic acid molecule according to the present disclosure comprises a polynucleotide encoding a polypeptide containing an amino acid sequence that is characterized by at least 85%, at least 86%, at least 87%, at least 88% at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with respect to any under SEQ ID NO: 1-38. In some embodiments, isolated nucleic acid molecules according to the present disclosure comprise a polynucleotide encoding a polypeptide containing an amino acid sequence characterized by 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with respect to any under SEQ ID NO: 1-38.

In accordance with some embodiments, the nucleic acid is contained in a vector, such as an expression vector. In accordance with some embodiments, the vector contains a promoter operably linked to the nucleic acid.

A number of promoters can be used to express the polypeptides described herein.

- 21 041568 document, including, without limitation, cytomegalovirus (CMV) intermediate early promoter, viral LTR such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 early promoter (SV40), E. coli lac UV5 and herpes simplex virus promoter tk. Regulated promoters may also be used. Such regulated promoters include promoters using the E. coli lac repressor as a transcriptional modulator to regulate transcription from promoters of lac operator-bearing mammalian cells [Brown, M. et al., Cell, 49:603-612 (1987)], promoters using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. sci. USA, 92:6522-6526 (1995)].

Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenolmurislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad. Can be used inducible promoters that include a repressor with an operon. In one embodiment, the Escherichia coli lac repressor can function as a transcriptional modulator to regulate transcription from promoters of mammalian lac operator-bearing cells [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992) [M. Gossen et al., Natl. Acad. sci. USA, 89:5547-5551 (1992)] combined a tetracycline repressor (tetR) with a transcription activator (VP 16) to create a mammalian tetR transcription activator fusion protein, tTa (tetR-VP 16), with a tetO-carrying minimal promoter derived from the major immediate early promoter of human cytomegalovirus (HCMV), with the creation of the tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used (Yao et al., Human Gene Therapy; Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

In addition, the vector may contain, for example, some or all of the following: a selectable marker gene, such as a neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the human CMV immediate-early gene for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; initiation of SV40 and ColE1 polyoma replication for proper episomal replication; internal ribosome binding sites (IRES), variable multiple cloning sites; as well as T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

An expression vector containing a nucleic acid can be transferred into a host cell by conventional techniques (eg, electroporation, liposomal transfection, and calcium phosphate precipitation), and then the transfected cells are cultured by conventional techniques to obtain the polypeptides described herein. In some embodiments, the expression of the polypeptides described herein is regulated by a constitutive, inducible, or tissue-specific promoter.

The host cells used to express the isolated polypeptides described herein can be either bacterial cells, such as Escherichia coli, or preferably eukaryotic cells. In particular, mammalian cells such as Chinese hamster ovary (CHO) cells, in combination with a vector such as the immediate early gene major promoter element from human cytomegalovirus, is an efficient expression system for immunoglobulins (Foecking et al. (1986), Powerful And Versatile Cockett et al (1990), High Level Expression Of Tissue Inhibitor Of Metalloproteinases In Chinese Hamster Ovary Cells Using Glutamine Synthetase Gene Amplification, Biotechnology, 8:662- 667). A number of expression vector host systems can be used to express the isolated polypeptides described herein. Such host expression systems provide carrier media by which the coding sequences for the isolated polypeptides described herein can be generated and then purified, as well as cells which, when transformed or transfected with suitable nucleotide coding sequences, can express the isolated polypeptides described in this document in situ. These include, without limitation, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing coding sequences for the isolated polypeptides described herein; yeast (eg, Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the isolated polypeptides described herein; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing sequences encoding the isolated polypeptides described herein; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid

- 22 041568 expression vectors (eg Ti plasmids) containing sequences encoding the isolated polypeptides described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphatic cells (see US Pat. No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell) containing recombinant expression constructs, containing promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from mammalian viruses (eg, adenovirus late promoter; vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors can be successfully selected depending on the application intended for the polypeptides to be expressed. For example, if a large amount of such a protein is required, then vectors that direct the expression of high levels of fusion protein products that are easily purified may be desirable to create pharmaceutical compositions of the polypeptides described herein. Such vectors include, but are not limited to, the E. coli pUR278 expression vector (Ruther et al. (1983), Easy Identification Of cDNA Clones, EMBO J. 2:1791-1794), wherein the coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is obtained; pIN vectors (Inouye et al. (1985), Up-Promoter Mutations In The lpp Gene Of Escherichia Coli, Nucleic Acids Res. 13:3101-3110; Van Heeke et al. (1989), Expression Of Human Asparagine Synthetase In Escherichia Coli , J. Biol. Chem. 24:5503-5509) and the like. pGEX vectors can also be used to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins. Typically, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione garose beads followed by elution in the presence of free glutathione.

The pGEX vectors are designed to include protease thrombin or factor Xa cleavage sites so that the cloned target gene product can be released from the GST fragment. The insect system uses the Autographa californica multiple nuclear polyhedrosis virus (AcNPV) as a vector for foreign gene expression. The virus grows in the cells of Spodoptera frugiperda. The coding sequence can be cloned individually into minor regions (eg the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg the polyhedrin promoter).

A number of virus-based expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the coding sequence of interest can be ligated into an adenovirus transcription/translation control complex, eg a late promoter and a three-component leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a minor region of the viral genome (eg, the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (see, for example, Logan et al. (1984), Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection, Proc. Natl. Acad. Sci. USA, 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG start codon and neighboring sequences. In addition, the start codon must be in-frame with the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic.

Expression efficiency can be enhanced by the inclusion of suitable transcriptional enhancer elements, transcription terminators, and the like. (See Bitter et al. (1987), Expression And Secretion Vectors For Yeast, Methods in Enzymol. 153:516-544). In addition, a host cell strain can be selected that modulates the expression of the inserted sequences, or modifies and processes the gene product in a particular desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. For example, in some embodiments, the polypeptides described herein may be expressed as a single gene product (e.g., as a single polypeptide chain, i.e., as a polyprotein precursor) requiring proteolytic cleavage by native or recombinant cellular machinery to form separate the polypeptides described herein.

The present disclosure thus encompasses the construction of a nucleic acid sequence for encoding a polyprotein precursor molecule comprising the polypeptides described herein, which includes coding sequences capable of directing post-translational cleavage of said polyprotein precursor. Post-translational cleavage of the polyprotein precursor results in the polypeptides described herein. Post-translational cleavage of a precursor molecule containing the polypeptides described herein may occur in vivo (i.e., in the host cell by native or recombinant cellular systems/mechanisms, e.g., furin cleavage at the appropriate site) or may occur in vitro (eg, incubation of said polypeptide chain in a composition containing proteases or peptidases of known activity, and/or in a composition containing conditions or reagents known to promote the desired proteolytic effect).

Purification and modification of recombinant proteins are well known in the art, so that the development of a polyprotein precursor may involve a number of options for implementation, not easily determined by the practitioner. Any known protease or peptidases known in the art can be used for the described modifications of the precursor molecule, for example thrombin or factor Xa (Nagai et al. (1985), Oxygen Binding Properties Of Human Mutant Hemoglobins Synthesized In Escherichia Coli, Proc. Nat. Acad. Sci. USA, 82:7252-7255, and reviewed in Jenny et al. (2003), A Critical Review Of The Methods For Cleavage Of Fusion Proteins With Thrombin And Factor Xa, Protein Expr. Purif. 31:1-11 , each of which is incorporated herein by reference in its entirety)), enterokinase (Collins-Racie et al. (1995), Production Of Recombinant Bovine Enterokinase Catalytic Subunit In Escherichia Coli Using The Novel Secretory Fusion Partner DsbA, Biotechnology, 13 :982-987, hereby incorporated by reference in its entirety)), furin, and AcTEV (Parks et al. (1994), Release Of Proteins And Peptides From Fusion Proteins Using A Recombinant Plant Virus Proteinase, A nal. Biochem. 216:413-417, hereby incorporated herein by reference in its entirety)), as well as FMDV protease C3.

Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells can be used, which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term production with high yield of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered that stably express the polypeptides described herein. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (eg, promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After the introduction of foreign DNA, it is possible to ensure the growth of engineered cells for 1-2 days in an enriched medium, and then switch to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow into foci, which in turn can be cloned and propagated into cell lines. This method can advantageously be used to construct cell lines that express the polypeptides described herein. Such engineered cell lines may be particularly useful in screening and evaluating polypeptides that interact directly or indirectly with the polypeptides described herein.

A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase genes (Wigler et al. (1977), Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Cultured Mouse Cells, Cell, 11:223-232), hypoxanthine-guanine phosphoribosyltransferases (Szybalska et al. (1992), Use Of The HPRT Gene And The HAT Selection Technique In DNA-Mediated Transformation Of Mammalian Cells First Steps Toward Developing Hybridoma Techniques And Gene Therapy, Bioessays, 14:495-500) and adenine phosphoribosyltransferases (Lowy et al. (1980), Isolation Of Transforming DNA: Cloning The Hamster aprt Gene, Cell, 22:817-823) can be used in tk cells^-, hgprt^- or aprt accordingly, antimetabolite resistance can also be used as a basis for selection for the following genes: dhfir, which confers resistance to methotrexate (Wigler et al. (1980), Transformation Of Mammalian Cells With An Amplifiable Dominant-Acting Gene, Proc. Natl. Acad. Sci. USA, 77:3567-3570; O'Hare et al. (1981), Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase, Proc. Natl. Acad. Sci. USA, 78: 1527-1531); gpt, which provides resistance to mycophenolic acid (Mulligan et al. (1981), Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase, Proc. Natl. Acad. Sci. USA, 78:2072-2076) ; neo that confers resistance to aminoglycosidase G-418 (Tolstoshev (1993), Gene Therapy, Concepts, Current Trials And Future Directions, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993), The Basic Science Of Gene Therapy, Science, 260:926-932 and Morgan et al.(1993), Human Gene Therapy, Ann. Rev. Biochem. 62:191217), and hygro, which confers resistance to hygromycin (Santerre et al. ) Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L

- 24 041568

Cells, Gene 30:147-156). Methods well known in the field of recombinant DNA technology and which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al. (1981), A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells, J. Mol. Biol. 150:1-14.

Expression levels of the polypeptides described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 ( Academic Press, New York, 1987) If a marker in a vector system expressing a polypeptide described herein is amplifiable, then increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. nucleotide sequence of a polypeptide as described herein or with a polypeptide as described herein, then production of the polypeptide will also be increased (Crouse et al. (1983), Expression And Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes, Mol. Cell. Biol. 3: 257-266).

Once a polypeptide described herein has been recombinantly expressed, it can be purified by any method known in the art for the purification of polypeptides, polyproteins, or antibodies (e.g., similar to antigen selectivity-based antibody purification schemes), e.g., by chromatography ( for example, ion exchange, affinity, especially by affinity for a specific antigen (optionally after selection of protein A if the polypeptide contains an Fc domain (or part thereof)) and column chromatography with size distribution), centrifugation, differential solubility, or using any other standard procedure for the purification of polypeptides or antibodies. Other aspects of the present disclosure relate to a cell containing the nucleic acid described herein, or the vector described herein.

The cell may be a prokaryotic or eukaryotic cell. In accordance with some embodiments, the cell is a mammalian cell. This document describes typical types of cells. Other aspects of the present disclosure relate to a cell expressing the modified BoNT polypeptides described herein. The cell may be a prokaryotic or eukaryotic cell. In accordance with some embodiments, the cell is a mammalian cell. This document describes typical types of cells. The cell may serve to propagate the nucleic acid, or to express the nucleic acid, or both. Such cells include, without limitation, prokaryotic cells, including, but not limited to, aerobic, microaerophilic, capnophilic, facultative, anaerobic, Gram-negative, and Gram-positive bacterial cell strains, such as those derived from, for example, Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis, Clostridia perfringens, Clostridia difficile, Caulobacter crescentus, Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls, Neisseria meningitidis, Pseudomonas fluorescens and Salmonella typhimurium; and eukaryotic cells, including but not limited to yeast strains such as, for example, those derived from Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Yarrowia lipolytica; insect cells and cell lines derived from insects, such as, for example, those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca sexta; and mammalian cells and cell lines derived from mammalian cells, such as, for example, those derived from mice, rats, hamsters, pigs, cattle, horses, primates, and humans. Cell lines can be obtained from the American Type Culture Collection, the European Cell Culture Collection, and the German Microorganism and Cell Culture Collection. Non-limiting examples of specific protocols for selecting, creating and using an appropriate cell line are described, for example, in INSECT CELL CULTURE ENGINEERING (Mattheus F. A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (J.M. Vlak et al. eds., Kluwer Academic Publishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUES OF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley and Sons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (Wiley-Liss, 4.sup.th ed. 2000); ANIMAL CELL CULTURE: A PRACTICAL APPROACH (John R.W. Masters ed., Oxford University Press, 3.sup.rd ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press, 2.sup.nd ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004).

These protocols are routine procedures within the scope of knowledge of a person skilled in the art and from the description given herein. Other aspects of the present disclosure relate to a method for producing a polypeptide described herein, wherein the method

- 25 041568 provides for obtaining the cell described herein, and the expression of the nucleic acid described herein in the specified cell. In accordance with some embodiments, the method further comprises isolating and purifying the polypeptide described herein.

In some embodiments, the botulinum neurotoxin can be obtained by creating and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermentable mixture according to known procedures. All botulinum toxin serotypes are initially synthesized as inactive single-stranded proteins that must be cleaved or torn apart by proteases to become neuroactive.

Bacterial strains that produce botulinum toxin serotypes A and G possess endogenous proteases and therefore serotypes A and G can be recovered from bacterial cultures predominantly in their active form. In contrast, botulinum toxin serotypes C, D, and E are synthesized by non-proteolytic strains and are therefore usually inactive when recovered from culture. Serotypes B and F are produced by both proteolytic and non-proteolytic strains and can therefore be recovered in active or inactive form. Proteolytic strains that produce, for example, the botulinum toxin type B serotype can degrade only a portion of the toxin produced. This document describes the production of BoNT/X polypeptides using these strains.

The exact ratio of broken and unbroken molecules depends on the duration of incubation and culture temperature. Therefore, a certain percentage of the drug, such as botulinum toxin type B, may be inactive. In one embodiment, the neurotoxin of the present disclosure is in an active state. In one embodiment, the neurotoxin is in an inactive state. In one embodiment, a combination of active and inactive neurotoxin is contemplated.

One aspect of the present disclosure relates to novel methods for producing BoNT using an in vitro transpeptidase reaction that ligates two non-toxic BoNT fragments. Such methods include the steps:

(i) obtaining a first BoNT fragment containing a light chain (LC) and an N-terminal heavy chain domain (H _N ), wherein the first BoNT fragment contains a C-terminal LPXTGG motif (SEQ ID NO: 60);

(ii) obtaining a second BoNT fragment containing the heavy chain C-terminal domain (H _C ); wherein the second BoNT fragment contains a specific protease cleavage site at its N-terminus;

(iii) cleavage of the second BoNT fragment with a specific protease, wherein the cleavage results in a free glycine residue at the N-terminus; and (iv) contacting the first BoNT fragment and the second BoNT fragment in the presence of a transpeptidase, thereby ligating the first BoNT fragment and the second BoNT fragment to form a ligated BoNT.

In some embodiments, the first BoNT fragment comprises an X-LC-H _n polypeptide as described herein fused to the C-terminal motif of LPXTGG (SEQ ID NO: 60) (e.g., SEQ ID NO: 45), or any of its options. In some embodiments, the second BoNT fragment comprises the HC polypeptide described herein or any variants thereof (eg, SEQ ID NO: 46). It should be appreciated that any BoNT fragments or domains can be ligated using the methods described herein.

The methods described herein can also be used to generate chimeric BoNTs. For example, the first BoNT fragment may be from BoNT serotype A, B, C, D, E, F, G, or X. Similarly, the second BoNT fragment may be from BoNT serotype A, B, C, D, E, F, G, or X. The person skilled in the art will be able to determine the combinations that can be made. In accordance with some embodiments, the chimeric BoNT polypeptides described herein (e.g., BoNT/X-LC-H _N -A1-H _C , BoNT/X-LC-H _N -B1-H _c , or BoNT/X-LC -H _n -C1-H _c ) is obtained using this method.

In some embodiments, the transpeptidase is a sortase. In some embodiments, sortase is derived from Staphylococcus aureus (SrtA).

Other peptide ligation systems available in the art can also be used to ligate two non-toxic BoNT fragments. For example, an intein-mediated protein ligation reaction provides for the ligation of a synthetic peptide or protein with an N-terminal cysteine residue to the C-terminus of a bacterially expressed protein via a native peptide bond (Evans et al, (1998), Protein Sci. 7, 2256-2264, Dawson et al., (1994), Science, 266, 776-779; Tam et al., (1995), Proc. Natl. Acad. Sci. USA, 92, 12485-12489, Muir et al., (1998), Proc Natl Acad Sci USA 95 6705-6710 Severinov and Muir (1998) J Biol Chem 273 16205-16209, the entire contents of which are incorporated herein by reference). Kits are commercially available (eg from New England Biolabs) for intein-mediated protein ligation reactions.

In accordance with some embodiments, the first BoNT fragment further comprises an affinity tag. In accordance with some embodiments, the affinity tag is fused to the first BoNT fragment at the N-terminus. In accordance with some embodiments, the affinity tag is fused to the first BoNT fragment at the C-terminus. In the case where the affinity tag is fused to the C-terminus of the first BoNT fragment, the transpeptidase cleaves between T and G in the LPXTGG motif (SEQ ID NO: 60) and removes the affinity tag before ligating the first BoNT fragment and the second BoNT fragment.

In accordance with some embodiments, the second BoNT fragment further comprises an affinity tag. In accordance with some embodiments, the affinity tag is fused to the first BoNT fragment at the N-terminus. In some embodiments, the affinity tag is fused to a second BoNT fragment at the C-terminus. In the case where the affinity tag is fused to the N-terminus of the first BoNT fragment, the transpeptidase cleaves at the specific protease cleavage site and removes the affinity tag before ligating the first BoNT fragment and the second BoNT fragment with the transpeptidase.

As used herein, the term affinity tag refers to a polypeptide sequence that can specifically bind to a substance or fragment, for example, a tag of six histidines specifically binds to Ni ²⁺ . Affinity tags can be added to proteins to facilitate their isolation. Affinity tags are typically fused to proteins using recombinant DNA techniques known to those skilled in the art. The use of affinity tags to facilitate protein isolation is also well known in the art. Suitable affinity tags that can be used in accordance with the present disclosure include, without limitation, His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Softag 1, Softag 3, TC, V5, VSV, Xpress, Halo and Fc.

The second BoNT fragment is cleaved by a specific protease at the N-terminus. Cleavage of the site with a specific protease results in a free glycine residue at the N-terminus of the second BoNT fragment. Suitable specific protease that can be used in accordance with the present disclosure includes, without limitation, thrombin, TEV, PreScission, enterokinase, and SUMO protease. In some embodiments, the specific protease is thrombin and the cleavage site is LVPR|GS (SEQ ID NO: 50).

The BoNT/X polypeptides described herein have potential for therapeutic applications. For example, BoNT/X may be more effective than other BoNT serotypes. BoNT/X is more variable and may be more effective in a wide range of cells due to its ability to cleave more substrates than other BoNT serotypes.

Thus, the present disclosure also covers pharmaceutical compositions containing BoNT/X polypeptides or chimeric molecules in accordance with the present disclosure. As will also become apparent from the present disclosure, the pharmaceutical composition in accordance with the present disclosure may additionally contain other therapeutic agents suitable for the particular disease for which the composition is intended to treat. In accordance with some embodiments, the pharmaceutical composition according to the present disclosure further comprises pharmaceutically acceptable carriers.

As used herein, the term pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition, or carrier medium such as a liquid or solid excipient, diluent, processing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or stearic acid). ) or solvent encapsulation material involved in the transfer or transport of a polypeptide from one site (eg, delivery site) of an organism to another site (eg, organ, tissue, or body part).

A pharmaceutically acceptable carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and harmless to the subject's tissue (eg, physiologically compatible, sterile, physiological pH, etc.). Some examples of materials that can serve as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) adjuvants such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyhydric alcohols such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free

- 27 041568 water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) a serum component such as serum albumin, HDL and LDL; (22) C ₂ -C ₁₂ alcohols such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Moisturizers, colorants, release agents, coatings, sweeteners, flavors, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as adjuvant, carrier, pharmaceutically acceptable carrier, or the like are used interchangeably herein. In some embodiments, the BoNT polypeptide of the present disclosure is administered in compositions by injection, via a catheter, via a suppository, or via an implant, wherein the implant is a porous, non-porous, or gel-like material, including a membrane such as a sialastic membrane, or fiber.

As a rule, when the composition is administered, materials are used that do not absorb the polypeptide in accordance with the present disclosure. According to other embodiments, the BoNT polypeptides of the present disclosure are delivered in a controlled release system. Such compositions and routes of administration are provided in US Patent Publication No. 2007/0020295, the contents of which are incorporated herein by reference. In one embodiment, a pump may be used (see, for example, Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). According to another embodiment, polymeric materials can be used (see, for example, Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance ( Smolen and Ball eds., Wiley, New York, 1984) Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science? 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Other controlled release systems are discussed in, for example, Langer, supra.

The BoNT polypeptides of the present disclosure may be administered as pharmaceutical compositions containing a therapeutically effective amount of a binding agent and one or more pharmaceutically compatible ingredients. According to exemplary embodiments, the pharmaceutical composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to a subject, such as a human.

Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the injection site. Typically, the ingredients are provided either separately or mixed together in a unit dosage form, for example as a dry lyophilized powder or anhydrous concentrate, in a sealed container such as an ampoule or sachet, indicating the amount of active agent. If the pharmaceutical composition is to be administered by infusion, it may be dispensed from an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration. The pharmaceutical composition for systemic administration may be a liquid, such as sterile saline, lactated Ringer's solution, or Hank's solution. In addition, the pharmaceutical composition may be in solid form and re-dissolved or suspended just prior to use. Freeze-dried forms are also contemplated. The pharmaceutical composition may be contained in a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles may be of any suitable structure, such as single layer or multilayer, as long as they contain compositions.

The polypeptides of the present disclosure can be captured in stabilized plasmid-lipid particles (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mole%) of cationic lipid, and stabilized with a polyethylene glycol (PEG) coating (Zhang Y.P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate or DOTAP are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, for example, US Pat. Nos. 4,880,635; 4906477; 4911928; 4917951; 4,920,016 and 4,921,757. The pharmaceutical compositions of the present disclosure may be administered or packaged as a unit dose, for example.

The term unit dose, when used in relation to a pharmaceutical composition in accordance with the present disclosure, refers to physically discrete units suitable as a unit dosage for a subject, with each unit containing a predetermined

- 28 041568 the amount of active material calculated to obtain the desired therapeutic effect, in association with the necessary diluent; those. carrier or carrier medium. In some embodiments, the BoNT/X polypeptides described herein may be conjugated to a therapeutic moiety, such as an antibiotic. Techniques for conjugating such therapeutic moieties to polypeptides, including, for example, Fc domains, are well known; see, for example, Amon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, p. 243-56, Alan R. Liss, Inc.); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery ( ^2nd Ed.), Robinson et al. (eds.), 1987, p. 623-53, Marcel Dekker, Inc.); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), 1985, p. 475-506); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 1985, p. 303-16, Academic Press; and Thorpe et al. (1982), The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev. 62:119-158. In addition, the pharmaceutical composition may be presented as a pharmaceutical kit containing (a) a container containing a polypeptide according to the present disclosure in lyophilized form, and (b) a second container containing a pharmaceutically acceptable diluent (eg, sterile water) for injection. A pharmaceutically acceptable diluent may be used to reconstitute or dilute the lyophilized polypeptide in accordance with the present disclosure. Optionally, such container(s) may be accompanied by a note in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, the note reflecting the authority's approval of the manufacture, use, or sale for human administration. In another aspect, an article of manufacture is provided that contains materials useful in the treatment of the diseases described above. In accordance with some embodiments, the product includes a container and a label.

Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a number of materials such as glass or plastic. In accordance with some embodiments, the container contains a composition that is effective for treating the disease described herein and can be sterile accessed. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is an isolated polypeptide according to the present disclosure. In some embodiments, the label on or with the container indicates that the composition is being used to treat the disease of choice. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, or dextrose solution. It may additionally include other materials desirable from a commercial and user point of view, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

BoNT polypeptides (eg, BoNT/X polypeptide), chimeric molecules and pharmaceutical compositions according to the present disclosure can be used to treat conditions associated with unwanted neuronal activities. Thus, further provided herein are methods of treating a condition associated with unwanted neuronal activity, the method comprising administering a therapeutically effective amount of a BoNT polypeptide, chimeric molecule, or pharmaceutical composition described herein, thereby treating the condition. In some embodiments, the BoNT polypeptides, chimeric molecules, and pharmaceutical compositions of the present disclosure are contacted with one or more neurons exhibiting unwanted neuronal activity.

Conditions that are typically treated with a neurotoxin (eg, skeletal muscle conditions, smooth muscle conditions, glandular conditions, neuromuscular disorder, autonomic disorder, pain, or aesthetic/cosmetic condition) are associated with unwanted neuronal activity, as defined by those skilled in the art. The introduction is carried out by bringing into contact an effective amount of the composition with neurons exhibiting undesirable activity. In accordance with some embodiments, the condition may be associated with overactive neurons or glands. Specific conditions contemplated for treatment by the methods discussed herein include, without limitation, spastic dysphonia, spasmodic torticollis, laryngeal muscle dystonia, oromandibular dystonia, lingual dystonia, cervical dystonia, focal arm dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disturbance, cerebral palsy, focal spasticity and other voice disorders, spastic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia and other disorders of muscle tone, as well as other disorders characterized by involuntary movements of muscle groups, lachrymation, hyperhidrosis, increased salivation, excessive gastrointestinal secretions, as well as other secretory disorders, pain due to muscle spasms, headache. In addition, the present disclosure can be used to treat dermatological or aesthetic/cosmetic conditions, such as reducing the frown line, reducing skin wrinkles.

One unique feature of the BoNT/X polypeptides of the present disclosure is their ability to cleave VAMP4, VAMP5 and Ykt6. Thus, the following describes the therapeutic use of BoNT/X polypeptides for conditions associated with unwanted secretion of activities in a wide range of cells. In accordance with some embodiments, the unwanted secretion is immune secretion. Conditions associated with unwanted immune secretion include, without limitation, inflammation, psoriasis, allergies, hemophagocytic lymphohistiocytosis, and alcoholic pancreatic disease.

The present disclosure may also be applied in the treatment of sports injuries. Borodic in US Patent No. 5,053,005 discloses methods for treating juvenile curvature of the spine, i. scoliosis using botulinum type A. The Borodic disclosure is incorporated herein by reference in its entirety. In one embodiment, using methods substantially similar to those disclosed by Borodic, a BoNT polypeptide can be administered to a mammal, preferably a human, for the treatment of curvature of the spine. According to a suitable embodiment, a BoNT polypeptide comprising type E botulinum fused to a leucine-based motif is administered. Even more preferably, a BoNT polypeptide comprising a leucine motif type A-E botulinum fused to the carboxyl terminus of its light chain is administered to a mammal, preferably a human, for the treatment of curvature of the spine.

In addition, BoNT polypeptides can be administered to treat neuromuscular disorders using well-known techniques that are commonly performed with botulinum type A. For example, the present disclosure can be used to treat pain, such as headache, pain due to muscle cramps, and various forms inflammatory pain. For example, Aoki US Pat. No. 5,721,215 and Aoki US Pat. No. 6,113,915 disclose methods of using botulinum toxin type A for the treatment of pain. The disclosures of these two patents are incorporated herein by reference in their entirety.

Autonomic nervous system disorders can also be treated with a modified neurotoxin. For example, a malfunction of the glands is a violation of the autonomic nervous system. Gland dysfunction includes excessive sweating and increased salivation. Respiratory dysfunction is another example of an autonomic nervous system disorder. Respiratory dysfunction includes chronic obstructive pulmonary disease and ASTHMA. Sanders et al. disclose methods of treating the autonomic nervous system; for example, the treatment of disorders of the autonomic nervous system, such as excessive sweating, increased salivation, asthma, etc., using naturally occurring botulinum toxins. The disclosure of Sander et al. incorporated herein by reference in its entirety.

In one embodiment, methods substantially similar to those of Sanders et al., but using a BoNT polypeptide, can be used to treat disorders of the autonomic nervous system such as those discussed above. For example, the BoNT polypeptide can be locally introduced into the nasal cavity of a mammal in an amount sufficient to generate autonomic nervous system cholinergic neurons that control mucus secretion in the nasal cavity. Pain that can be treated with a modified neurotoxin includes pain caused by muscle tension or spasm, or pain that is not associated with muscle spasm. For example, Binder discloses in US Pat. No. 5,714,468 that headache caused by vascular disorder, muscle tension, neuralgia, and neuropathy can be treated with a naturally occurring botulinum toxin, such as botulinum type A. Binder's disclosure is incorporated herein by reference in its entirety. .

In one embodiment, methods substantially similar to the Binder method, but using the BoNT polypeptide described herein, can be used to treat headache, especially those caused by vascular disorder, muscle tension, neuralgia, and neuropathy. Pain caused by muscle spasm can also be treated by administering the BoNT polypeptide described herein. For example, type E botulinum fused to a leucine-based motif, preferably at the carboxyl terminus of the botulinum type E light chain, can be injected intramuscularly at a pain/spasm site to relieve pain. In addition, the modified neurotoxin may be administered to a mammal to treat pain that is not associated with a muscle disorder such as spasm.

According to one broad embodiment, the methods of the present disclosure for treating non-spasm-related pain comprise central administration or peripheral administration of a BoNT polypeptide. For example, Foster et al. US Pat. No. 5,989,545 discloses that botulinum toxin conjugated to a targeting moiety can be administered centrally (intrathecally) to relieve pain. The disclosure of Foster et al. included in this document

- 30 041568 by reference in its entirety.

In one embodiment, methods substantially similar to those of Foster et al., but using the compositions described herein, can be used to treat pain. The pain to be treated may be acute pain or chronic pain. Acute or chronic pain that is not associated with muscle spasm can also be alleviated by local, peripheral administration of a modified neurotoxin at the site of actual or perceived pain in a mammal.

In one embodiment, the BoNT polypeptide is administered subcutaneously at or near a pain site, such as at or near a cut. In some embodiments, the modified neurotoxin is administered intramuscularly at or near a pain site, such as at or near a hematoma site, in a mammal. In some embodiments, the BoNT polypeptide is injected directly into a mammalian joint to treat or alleviate pain caused by arthritic conditions. Also, the frequently repeated injection or infusion of a modified neurotoxin at a site of peripheral pain falls within the scope of this disclosure. Routes of administration for such methods are known in the art and one of skill in the art can readily adapt them to the methods described herein (see, for example, Harrison's Principles of Internal Medicine (1998), edited by Anthony Fauci et al., 14 .sup.th edition, published by McGraw Hill).

As a non-limiting example, treatment of a neuromuscular disorder may include the step of locally administering an effective amount of the molecule to a muscle or group of muscles, treatment of an autonomic disorder may include the step of locally administering an effective amount of the molecule to a gland or glands, and treatment of pain may include the step of administering an effective amount of the molecule to the site of pain. . In addition, the treatment of pain may include the step of administering an effective amount of the modified neurotoxin to the spinal cord.

As used herein, the term therapeutically effective amount refers to the amount of each therapeutic agent according to the present disclosure required to provide a therapeutic effect in a subject, either alone or in combination with one or more other therapeutic agents. Effective amounts, as understood by those skilled in the art, will vary depending on the particular condition being treated, the severity of the condition, the individual parameters of the subject, including age, physical condition, size, sex and weight, duration of treatment, nature of concurrent therapy (if present), the specific route of administration, and similar factors within the knowledge and experience of the healthcare professional. These factors are well known to those skilled in the art and can be ascertained by no more than routine experimentation. As a rule, it is preferred that the maximum dose of the individual components or their combinations be used, i. e. the highest safe dose based on sound medical judgment. Those skilled in the art will appreciate, however, that a subject may require a lower dose or tolerated dose for medical reasons, psychological reasons, or virtually any other reason. Empirical considerations such as half-life will generally guide dosage determination. For example, therapeutic agents that are compatible with the human immune system, such as polypeptides containing regions from humanized antibodies or fully human antibodies, can be used to extend the half-life of the polypeptide and to prevent the host's immune system from attacking the polypeptide.

The frequency of administration may be determined and adjusted during the course of therapy and is generally, but not necessarily, based on treatment and/or suppression and/or attenuation and/or delay of the disease. Alternatively, sustained sustained release formulations of the polypeptide may be suitable. Various formulations and devices are known in the art to achieve sustained release. In some embodiments, the dose is administered daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, the dosage frequency is once a week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, or every 3 months, or less frequently. The progress of this therapy is easily controlled by traditional methods and analyses.

The dosage regimen (including the polypeptide employed) may vary over time. In some embodiments, doses ranging from about 0.01 to 1000 mg/kg may be administered to a normal weight adult subject. In some embodiments, the dose is from 1 to 200 mg. The specific dosing regimen, ie. the dose, time, and repeatability will depend on the particular subject and that subject's medical history, as well as on the properties of the polypeptide (such as the half-life of the polypeptide and other factors well known in the art).

For the purposes of this disclosure, the appropriate dosage of a therapeutic agent described herein will depend on the specific agent (or compositions) used, composition and route of administration, type and severity of disease, administration of the polypeptide for preventive or therapeutic purposes, prior therapy , the subject's medical history and response to the antagonist, as well as the opinion of the attending physician. Typically, a physician will generally administer the polypeptide until a dosage is reached that provides the desired result.

Administration of one or more polypeptides may be continuous or discontinuous, depending on, for example, the physiological state of the recipient, the therapeutic or prophylactic purpose of administration, and other factors known to those skilled in the art. Administration of the polypeptide may be substantially continuous over a preselected period of time, or may be in divided dose series, eg, either before, during, or after disease progression. As used herein, the term treatment refers to the use or administration of a polypeptide or a composition comprising a polypeptide to a subject in need thereof.

The term "subject" optionally refers to an individual who has a disease, symptom of a disease, or predisposition to a disease requiring treatment, cure, alleviation, amelioration, change, cure, elimination, amelioration, or effect on the course of a disease, symptom of a disease, or predisposition to a disease. In accordance with some embodiments, the subject has a CDI. In accordance with some embodiments, the subject has cancer. In accordance with some embodiments, the subject is a mammal. In accordance with some embodiments, the subject is a non-human primate. In accordance with some embodiments, the subject is a human. Relief of a disease includes delaying the development or progression of a disease or reducing the severity of a disease. Relief of the disease does not require mandatory treatment results.

As used herein, the term delaying the development of a disease means delaying, inhibiting, slowing down, postponing, stabilizing and/or delaying the progression of a disease. Such a delay may be of varying length depending on the history of the disease and/or the individuals being treated. A method that slows or alleviates the development of a disease or slows the manifestation of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease in a given time frame and/or reduces the severity of symptoms in a given time frame compared to a situation without using the method. Such comparisons are usually based on clinical studies using a sufficient number of subjects to obtain a statistically significant result.

The term development or progression of a disease refers to the initial manifestations and/or subsequent progression of a disease. Disease progression can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression, which may be undetected. For the purposes of this disclosure, development or progression refers to the biological course of symptoms. The term development includes occurrence, recurrence, and onset.

As used herein, the term onset or onset of a disease includes initial onset and/or relapse. Conventional methods known to those of skill in the medical field may be used to administer the isolated polypeptide or pharmaceutical composition to a subject, depending on the type of disease being treated or the site of the disease. The composition may also be administered by other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir.

As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, this may include administration to the subject via depot injection routes, for example using a 1-, 3-, or 6-month depot injection or biodegradable materials and methods.

As used herein, the term subject refers to a human or animal. Typically, the animal is an invertebrate animal such as a primate, rodent, pet, or companion animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and rhesus macaques. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Domestic and companion animals include cows, horses, pigs, deer, bison, buffalo, cat species such as domestic cat, dog species such as dog, fox, wolf, bird species such as chicken, emu, ostrich, and fish, such as trout, catfish and salmon. The patient or subject includes any subgroup of the foregoing, such as all of those mentioned above, except for one or more groups or one or more species, such as humans, primates, or rodents. In accordance with some embodiments of the aspects described in this

- 32 041568 document, the subject is a mammal, such as a primate, such as a human.

The terms patient and subject are used interchangeably herein. The subject may be male or female. The subject may be a fully developed subject (eg, an adult) or a subject in the process of development (eg, a child, infant, or fetus). Preferably the subject is a mammal. The mammal may be a human, non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, without being limited to these examples. Mammals other than humans can be successfully used as subjects that represent animal models of disorders associated with unwanted neuronal activity. In addition, the methods and compositions described herein can be used to treat pets and/or companion animals.

The following examples are intended to illustrate some embodiments, but not to limit. The entire contents of all referenced documents (including but not limited to literature references, issued patents, published patent applications and pending patent applications) cited herein are hereby incorporated by reference herein.

Examples

Table 1

BoNT polypeptide sequences

SEQ ID NO: Description Subsequence 1 BoNT/X wt MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKrovroKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWI KHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY

- 33 041568

NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWY 2 BoNT/X wt LC-Hn MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIK VLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AF D IN DIPF SEFDDLINQ YKNEI 3 BoNT/X wt LC MKLEINKFNYNDProGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIK VLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKN 4 BoNT/X MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK

- 34 041568

wt C461S NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIK VLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPSSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IY VDKLTTFHFLE AQN IDES IDS SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYI<ALSRQANAIKMNMEFQLANYI<GNIDDI< AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWY LRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRI<HTI<TNWYFIPI<DEGWDED 5 BoNT/X wt C461A MI<LEINI<FNYNDPIDGINVITMRPPRHSDI<INI<GI<GPFI<AFQVII< NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIK VLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE

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RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPASLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWYFIPKDEGWDED 6 BoNT/X wt C467S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGSIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL

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VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWYFIPKDEGWDED 7 BoNT/X wt C467A MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGAIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWI KHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD

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HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWYFIPKDEGWDED 8 BoNT/X wt C1240S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSffiTGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFW IKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYSQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWYFIPKDEGWDED

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9 BoNT/X wt C1240S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWI KHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYAQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWYFIPKDEGWDED 10 BoNT/X wt C461S/C1 240A MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV

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QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPSSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYAQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKH TKTNWYFIPKDEGWDED eleven BoNT/X wt C461S/C1 240S MKLEINKFNYNDProGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPSSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS

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DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYSQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRI<HTI<TNWYFIPI<DEGWDED 12 BoNT/X wt C461A/C1 240S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPASLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI

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SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYSQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRI<HTI<TNWYFIPI<DEGWDED 13 BoNT/X wt C461A/C1 240A MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPASLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWI KHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYAQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW

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YFQNYENLNLRI<HTI<TNWYFIPI<DEGWDED 14 BoNT/X wt C467S/C1 240A MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGSIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWI KHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYAQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRI<HTI<TNWYFIPI<DEGWDED 15 BoNT/X wt C467S/C1 240S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID

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SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGSIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYSQL KTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWYFIPKDEGWDED 16 BoNT/X wt C467A/C1 240S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGAIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS

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PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYSQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRKHTKTNWYFIPKDEGWDED 17 BoNT/X wt C467A/C1 240A MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGAIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTS

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DINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSI SFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRD HNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISS IWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLY NYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSF GYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNK DFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQ EKYRNYAQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAW YFQNYENLNLRI<HTI<TNWYFIPI<DEGWDED 18 BoNT/X WT LC-H _N C461A MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPASLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPF SEFDDLINQ YI< NE I 19 BoNT/X wt LC-Hn C461S MKLEINKFNYNDProGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV

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QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPSSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYI<ALSRQANAII<MNMEFQLANYI<GNIDDI< AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AF D IN DIPF SEFDDLINQ YKNEI 20 BoNT/X wt LC-Hn C467A MI<LEINI<FNYNDPIDGINVITMRPPRHSDI<INI<GI<GPFI<AFQVII< NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGAIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AF D IN D IPF SEFDDLINQ YKNEI 21 BoNT/X wt LC-Hn MI<LEINI<FNYNDPIDGINVITMRPPRHSDI<INI<GI<GPFI<AFQVII< NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA

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C467S YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGSIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AF D IN DIPF SEFDDLINQ YKNEI 22 BoNT/X- LC-Hn- Al-Hc MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI

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AFDINDIPFSEFDDLINQYKNEIIINTSILNLRYESNHLIDLSRYASK INIGSKVNFDPIDKNQIQLFNLES SKIEVILKNAIVYNSMYENF STS FWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDT QEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLI DQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNE KEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYV DVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYAS GNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEI PDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQ FNNIAKL VASNW YNRQIERS SRTLGC S WEFIP VDDGWGERPL 23 BoNT/X- LC-Hn- Bl-Hc MI<LEINI<FNYNDPIDGINVITMRPPRHSDI<INI<GI<GPFI<AFQVII< NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIK VLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IY VDKLTTFHFLE AQN IDES IDS SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYI<ALSRQANAII<MNMEFQLANYI <GNIDDI< AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIILNNIILNLRYKDNNLIDLSGYGA KVE VYDGVELNDKNQFKLT S S ANSKIRVTQN QNIIFNS VFLDF S V SFWIRIPI<YI<NDGIQNYIHNEYTIINCMI<NNSGWI<ISIRGNRIIWT LIDINGI<TI<SVFFEYNIREDISEYINRWFFVTITNNLNNAI<IYINGI< LESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQ SNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIK LKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSIN

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DDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDS DEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGI VFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE 24 BoNT/X- LC-Hn- Cl-Hc MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGIT STYIF YQWLRSIVKDF SDETGKID VIDKS S DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIINDSKILSLQNRKNTLVDTSGYN AEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMY E SFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGIISNFLVFTLK QNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIYIN GKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRD FYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMV NIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRGNTNDT RVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGL REQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTY RFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE 25 BoNT/X- LC-Hn- Al-Hc C461S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS

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EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPSSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIIINTSILNLRYESNHLIDLSRYASK INIGSKVNFDPIDKNQIQLFNLES SKIEVILKNAIVYNSMYENF STS FWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDT QEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLI DQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNE KEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYV DVNNVG IRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYAS GNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEI PDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQ FNNIAKL VASNW YNRQIERS SRTLGC S WEFIP VDDGWGERPL 26 BoNT/X- LC-Hn- Bl-Hc C461S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEF LQGVIKVLERIKSKPEGEKLLELIS S SIPLPLVSNGALTLSDNETIA YQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLS EVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHE LVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGID SKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPV QGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKE RPIDPIYVNILDDNS YSTLEGFNIS SQGSNDFQGQLLES S YFEKIES NALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVS YPSSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKD

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KLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKT IYVDKLTTFHFLEAQNIDESID S SKIRVELTD S VDEALSNPNK VYS PFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSS DTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPIL VGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWW GTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDK AKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKV QDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRLNKNI AFDINDIPFSEFDDLINQYKNEIILNNIILNLRYKDNNLIDLSGYGA KVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSV SFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWT LIDINGI<TI<SVFFEYNIREDISEYINRWFFVTITNNLNNAI<IYINGI< LESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQ SNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIK LKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSIN DDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDS DEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGI VFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

BoNT/XLC-Hn-C1MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE

Hc C461S

FLQGVIK VLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH

ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP

VQGRRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN

VSYPSSLLNGCIEVENKDLFLISNKDSLNDINLSEEKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDELHGA

TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA

QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI

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PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIINDSKILSLQNRKNTLVDTS GYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVY NSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGIISNFL VFTLKQNEDSEQS INF S YDISNN APGYNK WFF VTVTNNMMGNM KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINM WIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKE YYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRG NTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDI YAIGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGI CSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFV PVSE 28 BoNT/X- LC-Hn-A1- Hc C467S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGSIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEniNTSILNLRYESNHLIDLSR YASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYE NFSTSFWIRIPKYFNS ISLNNEYTIINCMENNSGWKVSLNYGEIIW TLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYI NGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFD

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KELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDP NKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIK KYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKIL SALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGF IGFHQFNNIAKLVASNWYNRG SW 29 BoNT/X- LC-H _N -B1- Hc C467S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGSIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNmESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIILNNIILNLRYKDNNLIDLS GYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVF LDFSVSFWIRIPKYKNDGI QNYIHNEYTIINCMKNNSGWKISIRG NRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNA KIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSI FNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAG NKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRR KSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKL FLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGI HRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIP

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KDEGWTE thirty BoNT/X- LC-Hn-C1- Hc C467S MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGSIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIINDSKILSLQNRKNTLVDTS GYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVY NSMYESFSISFWIRINKW VSNLPGYTIIDSVKNNSGWSIGIISNFL VFTLKQNEDSEQS INF S YDISNN APGYNK WFF VTVTNNMMGNM KIYINGKLmTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINM WIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKE YYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRG NTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDI YAIGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGI CSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFV PVSE 31 BoNT/X R360A/Y36 3F MKLEINKFNYNDProGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGLMEGTL SEVSFSPFYLKPFVDESYGNYRSLVFHRE

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ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVAKHFL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLS GTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSAT DNFSISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLI WYLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKI EEKDIS SIWNTEVDDPIIFRLKNNRDTQ AFTLLDQF SIYRKELNQN EVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIR EYWSSFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLD GLVRNKDFIQLEIDGYNMGI SADRFNEDTNYIGTTYGTTHDLTT DFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRD WVYS S AWYFQNYENLNLRKHTKTNWYFIPKDEGWDED 32 BoNT/X H227Y MKLEINKFNYNDProGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIK VLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMY ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI

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KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLS GTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSAT DNFSISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLI WYLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKI EEKDIS SIWNTEVDDPIIFRLKNNRDTQ AFTLLDQF SIYRKELNQN EVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIR EYWSSFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLD GLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTT DFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRD WVYS S A W YFQN YENLNLRK HTKTN W YFIPKDEGWDED 33 BoNT/X E228Q MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH QLVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGG IDSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL

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NKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLS GTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSAT DNFSISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLI WYLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKI EEKDIS SIWNTEVDDPIIFRLKNNRDTQ AFTLLDQF SIYRKELNQN EVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIR EYWSSFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLD GLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTT DFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRD WVYS S AWYFQNYENLNLRKHTKTNW YFIPKDEGWDED 34 BoNT/X H231Y MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH ELVYVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLS GTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSAT DNFSISFWIKHPKPTNL AFTLLDQF SIYRKELNQN EVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIR

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GLVRNKDFIQLEroGYNMGISADRFNEDTNYIGTTYGTTHDLTT DFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRD WVYS S AWYFQNYENLNLRKHTKTNW YFIPKDEGWDED 35 BoNT/X- LC-Hn R360A/Y36 3F MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVAKHFL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPF SEFDDLINQ YKNEI 36 BoNT/X- LC-Hn H227Y MKLEINKFNYNDProGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMY ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV

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YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPF SEFDDLINQ YKNEI 37 BoNT/X- LC-Hn E228Q MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH QLVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGG IDSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPF SEFDDLINQ YKNEI 38 BoNT/X- LC-Hn H231Y MKLEINKFNYNDProGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH ELVYVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI

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ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPF SEFDDLINQ YKNEI 39 VAMP1 MSAPAQPPAEGTEGTAPGGGPPGPPPNMTSNRRLQQTQAQVEE WDIIRVNVDKVLERDQKLSELDDRADALQAGASQFESSAAKL KRKYWWKNCKMMMIMLGAICAIIVVVIVIYFFT 40 VAMP2 MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVV DIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKR KYWWKNLKMMIILGVIC AIILIIIIVYF S S 41 VAMP3 MSTGVPSGSSAATGSNRRLQQTQNQVDEVVDIMRVNVDKVLE RDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNCKMW AIGISVLVIIVIIIIVWCVS 42 VAMP4 MPPKFKRHLNDDDVTGSVKSERRNLLEDDSDEEEDFFLRGPSGP RFGPRNDKIKHVQNQVDEVIDVMQENITKVIERGERLDELQDKS ESLSDNATAFSNRSKQLRRQMWWRGCKIKAIMALVAAILLLVIII LIVMKYRT 43 VAMP5 MAGIELERCQQQANEVTEIMRNNFGKVLERGVKLAELQQRSDQ LLDMSSTFNKTTQNLAQKKCWENIRYRICVGLVVVGVLLIILIVL LVVFLPQ S SD S S S APRTQD AGIASGPGN 44 Ykt6 VAFTLLEKVLDEFSKQVDRIDWPVGSPATIHYPALDGHLSRYQN PREADPMTKVQAELDETKIILHNTMESLLERGEKLDDLVSKSEV LGTQSKAFYKTARKQNSCCAII 45 BoNT/X- LC-Hn- LPETGG MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIK NIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDE FLQGVIKVLERIKSKPEGEKLLELIS S SIPLPL VSNGALTLSDNETI AYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTL

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SEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMH ELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGI DSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIP VQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYL KERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKI ESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTN VSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFF KDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEI KTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKV YSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVID KSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEF TIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKA QWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMI PKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLS S SLRRKVSIRL NKNIAFDINDIPF SEFDDLINQYKNEILPETGG 46 G-BoNT/X- hc GEDYEVLNLGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKI KGSENSTIKIAMNKYLRFSATDNFSISFWIKHPKPTNLLNNGIEYT LVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYIAFNGWN LITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRD TQAFTLLDQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQ YNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSDSKTITFPNNIR YGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMGISADRF NEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHK SGLMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTN WYFIPKDEGWDED 47 BoNT/Al- hc IINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNL ES SKIEVILKNAIVYNSMYENF STSFWIRIPK YFNSISLNNEYTIIN CMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDY INRWIFVTITNNRLNNSI<IYINGRLIDQI<PISNLGNIHASNNIMFI< LDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDI<PYYMLNLYDPNI<YVDVNNVGIRGYMYLI<GPRG SVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVV VKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKND QGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQI ERSSRTLGCSWEFIPVDDGWGERPL

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48 BoNT/Bl- hc ILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTS SANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNE YTIINCMKNNSGWKISIRGNRIIWTLroiNGKTKSVFFEYNIREDIS EYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFK LDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFW GNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNS KYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQE WRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQ LLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVK RKPYNLKLGCNWQFIPKDEGWTE 49 BoNT/Cl- hc INDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLG SSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYT IIDS VKNNSGWSIGIISNFLVFTLKQNED SEQ SINF S YDISNNAPGY NKWFFVTVTNNMMGNMKIYINGKLIDTIKVKELTGINFSKTITFE INKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINILFNSLQYT NVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTR RNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYN LFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTY YYASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVK QGNYASLLESTSTHWGFVPVSE 50 Site split AND I thrombin LVPRGS 51 TEV ENLYFQG 52 Website split AND I Pre Seis si on LEVLFQGP 53 Site split IA factor Ha IEGR 54 Website split IDGR

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IA factor Ha 55 Site cleaved by enterokin ZOY DDDDK 56 Site cleaved by SUMO protease AHREQIGG

* Mutations are underlined.

New botulinum neurotoxin and its derivatives.

Botulinum neurotoxins (BoNTs) are among the most dangerous potential bioterrorism agents and are also being used clinically to treat an increasing list of medical conditions. There are seven serotypes of BoNT (BoNT/A-G) currently known, but no new types have been identified in the past 45 years. A search of the genomic database of Clostridium botulinum strains revealed a new type of BoNT named BoNT/X. This toxin showed the lowest sequence identity to other BoNTs and was not recognized by antisera raised against known BoNT types. It cleaves vesicle-associated membrane protein (VAMP) in neurons, which is also a target for BoNT/B/D/F/G, but BoNT/X cleaves at a site (between Arg66-Ala67 in VAMP2) that is unique to this toxin. To confirm BoNT/X activity, a limited amount of full length BoNT/X was collected by covalently linking two non-toxic BoNT/X fragments using transpeptidase (sortase). The harvested BoNT/X penetrated cultured neurons, cleaved VAMP2, and caused atrophic paralysis in mice as measured by finger abduction index analysis. Together, these data established BoNT/X as a new type of BoNT. Its identification presents an urgent challenge for the development of effective countermeasures, and also represents a new tool for potential therapeutic applications.

A search of genomic databases revealed a new BoNT gene.

In an attempt to explore the evolutionary landscape of BoNTs, iterative Hidden Markov model searches were performed on the PubMed sequence database using seven BoNT sequences as probes. The search successfully identified the major serotypes, subtypes, and mosaic toxins of BoNT, as well as the related tetanus neurotoxin (TeNT) (Fig. 5). Surprisingly, a novel BoNT gene (GenBank No. BAQ12790.1) was also identified from the recently reported genomic sequence of Clostridium botulinum strain 111. This toxin gene is referred to herein as BoNT/X.

Phylogenetic analysis revealed that BoNT/X is clearly distinct from all other BoNTs and TeNTs (Fig. 1A). It has the lowest protein sequence identity (<31%) of any other BoNT among pairwise comparisons in the BoNT/TeNT family (FIG. 1A). For example, BoNT/A and BoNT/B share 39% sequence identity, and BoNT/B and BoNT/G share 58% sequence identity. In addition, a sliding window of sequence comparisons showed that low degree similarity was evenly distributed across the BoNT/X sequence compared to the other BoNT family and TeNT (FIG. 1B), indicating that this is not a mosaic toxin.

Despite low sequence identity, overall domain arrangement and some key features of BoNT appear to be conserved in BoNT/X (Fig. 1B), including: (1) a conserved zinc-dependent HExxH protease motif (residues 227-231 , HELVH (SEQ ID NO: 92)) is in the putative LC; (2) at the interface between putative LC and HC are two conserved cysteines that can form an important interchain disulfide bond; (3) a conserved SxWY receptor binding motif exists in the putative HC (residues 1274-1277, SAWY (SEQ ID NO: 93)), which recognizes lipid co-receptor gangliosides ^{43, 44} .

As expected, the BoNT/X gene is preceded by the putative NTNHA gene (FIG. 1C). They are located in the OrfX gene cluster. However, the OrfX gene cluster in BoNT/X has two unique features compared to the other two known OrfX clusters (Fig. 1C): (1) there is an additional OrfX2 protein (designated OrfX2b) adjacent to the BoNT/X gene that is not known. reported for any other OrfX clusters; (2) the reading frame of the OrfX genes has the same direction as

- 64 041568 as the BoNT/X gene, while they are usually opposite to the direction of the BoNT gene in other OrfX clusters (Fig. 1C). Together, these features indicate that BoNT/X may constitute a unique evolutionary branch of the BoNT family.

LC BoNT/X cleaves VAMP2 at a new site.

Next, it was checked whether BoNT/X is a functional toxin. First investigated LC BoNT/X (X-LC). The LC boundary (residues 1-439) was determined by sequence alignment with other BoNTs. Synthesized cDNA encoding LC and received LC as His6-tagged recombinant protein in E. coli. X-LC was incubated with extracts in rat brain surfactant (BDE) and immunoblotting analysis was used to examine the cleavage of the three dominant SNARE proteins in the brain - SNAP-25, VAMP2 and syntaxin 1. LC BoNT/A (A-LC) and BoNT/B (B-LC) were analyzed in parallel as controls. Cleavage of SNAP-25 with BoNT/A creates a smaller fragment that can still be recognized by immunoblot, while digestion of VAMP2 with BoNT/B nullifies the VAMP2 signal on immunoblot (Fig. 2A). Synaptophysin (Syp), a synaptic vesicular protein, was also identified as an internal loading control. Incubation of X-LC with rat brain DTE did not affect syntaxin 1 or SNAP-25, but abolished VAMP2 signals (Fig. 2A). LC BoNTs are zinc dependent proteases ²⁵ . As expected, EDTA prevented the cleavage of SNARE proteins by X-, A- and B-LC (FIG. 2A). To further confirm that X-LC cleaves VAMP2, the cytosolic domain of VAMP2 (residues 1-96) was purified as a His6-tagged protein. Incubation of VAMP2 (1-96) with X-LC converted the VAMP2 band into two lower molecular weight bands on an SDS-PAGE gel (FIG. 2B), confirming that X-LC cleaves VAMP2.

To identify the cleavage site in VAMP2, VAMP2 protein (1-96) was analyzed with or without pre-incubation with X-LC by liquid chromatography with tandem mass spectrometry (LC-MS/MS, Fig. 2C-2E, see details). below). A single peak of the dominant peptide appeared after incubation with X-LC (FIGS. 2C, 2E and 6). Its molecular weight was determined to be 3081.7, which only corresponds to the peptide sequence of residues A67-L96 of VAMP2 (FIGS. 2C, 2E). Accordingly, another fragment from the start of the His6 tag to the rest of VAMP2 R66 was also detected (FIG. 2D). To further confirm this result, the assay was repeated with another VAMP2 fragment, GST-labeled recombinant VAMP2 (33-86) (FIG. 7). Incubation with X-LC gave one dominant peak at molecular weight 2063.1, which only corresponds to the peptide sequence of residues A67-R86 of VAMP2 (Fig. 7D, 7E). As expected, another fragment was also detected from the start of the GST tag to the rest of VAMP2 R66 (FIG. 7F). Together, these results demonstrate that X-LC has a single cleavage site in VAMP2 between R66 and A67.

R66-A67 is a new cleavage site, different from the established target sites for all other BoNTs (FIG. 2F). This is also the only BoNT cleavage site located in the region previously known as the SNARE motif (Fig. 2F, shaded areas) ⁴⁵ . The VAMP family of proteins includes VAMP1, 2, 3, 4, 5, 7, 8, as well as related Sec22b and Ykt6. R66-A67 is conserved in VAMP1 and VAMP3, which are highly homologous to VAMP2, but not in other VAMP homologues such as VAMP7 and VAMP8. To confirm X-LC specificity, full length HA-labeled VAMP1, 3, 7, 8 and myc-labeled Sec22b and Ykt6 were expressed in HEK293 cells by transient transfection. Cell lysates were incubated with X-LC (Fig. 2G). VAMP1 and 3 were digested with X-LC, as evidenced by a shift in the immunoblot signal to lower molecular weight, while VAMP7, VAMP8 and Sec22B were resistant to X-LC (Fig. 2G).

Surprisingly, Ykt6 was cleaved with X-LC (FIG. 2G). This result was confirmed using a purified GST-labeled Ykt6 fragment that shifted to a lower molecular weight band after incubation with X-LC (FIG. 2H). The cleavage site was determined as K173-S174 by mass spectrometry analysis of intact Ykt6 versus X-LC digested Ykt6 (FIG. 13A). This is a homologous site to the cleavage site in VAMP2 (FIG. 2F), indicating that the location of the cleavage site is conserved across VAMPs. Among VAMP members, VAMP4 contains the same pair of residues (K87-S88) at this site as Ykt6. The GST-labeled cytoplasmic domain of VAMP4 was found to be efficiently cleaved by X-LC (FIG. 2I). Accordingly, X-LC cleaved native VAMP4 in BDE (FIG. 4J). As a control, Sec22b was not digested with X-LC in BDE. In addition, the GST-tagged cytoplasmic domain of VAMP5 was also cleaved, albeit at a slower rate than VAMP2 and VAMP4 (Fig. 2I). Cleavage sites were confirmed as K87-S88 in VAMP4 and R40-S41 in VAMP5 by mass spectrometry analysis (FIG. 14). Both are site homologous to the cleavage site in VAMP2 (FIG. 2F). The ability of X-LC to cleave VAMP4, VAMP5, and Ykt6 is highly unusual, as their sequences differ significantly from VAMP 1/2/3. BoNT/X is the first BoNT capable of cleaving VAMP outside the canonical targets of VAMP1/2/3 ⁶⁶ . X-LC also cleaved VAMP4 in BDE and cleavage was blocked with EDTA (FIG. 2J).

A notable feature of BoNT/X is its unique ability to cleave VAMP4 and Ykt6. VAMP4 is widely expressed and is known to mediate vesicle fusion between the network

- 65 041568 trans-Golgi (TGN) and endosomes, as well as homotypic fusion of endosomes ^{59, 60} . Ykt6 is an atypical SNARE without a transmembrane domain ^67-70 . It anchors in membranes via lipidation, which allows its membrane association to be dynamically regulated. Ykt6 is an essential protein in yeast, participating in multiple membrane fusion events, including ER-Golgi, intra-Golgi, endosome-Golgi-vacuolar, and autophagosomal formation. Its function in mammalian cells has not yet been established. Traditionally, BoNTs have been known to be limited to SNARE targets that mediate vesicular exocytosis at plasma membranes. BoNT/X is the first BoNT that is able to cleave SNARE, mediating various intracellular membrane migration events.

Interestingly, both VAMP4 and Ykt6 are upregulated in neurons. Recent studies have shown that VAMP4 may also contribute to asynchronous synaptic vesicular exocytosis, growthosomal exocytosis, and activity-dependent volumetric endocytosis (ADBE) in neurons ^61-63 . The role of Ykt6 in neurons remains to be established, but it has been shown to suppress α-synuclein toxicity in models of Parkinson's disease ^{71, 72} . Another BoNT/X substrate, VAMP5, is mainly expressed in muscle cells and its function has not yet been established ⁶⁴ . BoNT/X will be a powerful tool to investigate the functions of VAMP4, Ykt6, and VAMP5, as well as their associated membrane migration events. In addition, VAMP4 is involved in the release of granules in immune cells ⁶⁵ , thus BoNT/X may have a unique potential among all BoNTs to modulate inflammatory secretion in immune cells.

Proteolytic activation of BoNT/X.

BoNT is initially produced as a single polypeptide. The linker site between LC and H _N must be cleaved by either bacterial or host proteases in a process known as activation, which is important for BoNT activity. The LC and _HN in BoNT remain connected by an interstrand disulfide bond until the LC translocation into the cytosol of the cells, where the disulfide bond is repaired to release the LC into the cytosol. Sequence alignment showed that BoNT/X contained the longest linker region between two conserved cysteines compared to all other BoNTs (C423-C467, Fig. 3A). In addition, the BoNT/X linker site contains an additional cysteine (C461) that is unique to BoNT/X.

To test whether the linker site between LC and H _N in BoNT/X is susceptible to proteolytic cleavage, a recombinant X-LC-H _N fragment (residues 1-891) in E. coli was prepared and subjected to limited proteolysis with Lys- C, which cuts along the C-terminal side of the lysine residues. To identify a susceptible cleavage site under conditions of limited proteolysis, X-LC-H _N was analyzed using Tandem Mass Tag (TMT) and a tandem mass spectrometry approach. TMT tags do not have an N-terminus (and lysines). Limited proteolysis with Lys-C gives additional free N-termini that cannot exist in an intact X-LC-HN sample (see below for details). Briefly, intact X-LC-HN samples were labeled with mild TMT and an equal number of X-LC-HN samples were subjected to Lys-C followed by heavy TMT labeling. Then both samples were digested with chymotrypsin, combined together and subjected to quantitative analysis by mass spectrometry. The list of identified peptides is shown in table. 2. The light TMT:heavy TMT ratio was typically 2-fold relative to each other for each peptide, except for five peptides starting with N439 that showed no signal for light TMT labeling, indicating that this is a new N-terminus created by cutting Lys-C (Fig. 3A, Table 2). Thus, Lys-C preferentially cuts K438-N439 under limited proteolytic conditions, demonstrating that the linker site is protease sensitive (FIG. 3A).

The importance of this proteolytic activation for BoNT/X function was further tested. It has previously been shown that incubation of high concentrations of LC-H _N BoNT with cultured neurons resulted in LC-HN infiltration into neurons, probably due to non-specific uptake in neurons ^{46, 47} . Using this approach, the efficacy of intact and activated X-LC-HN was compared in cultured rat cortical neurons. Neurons were subjected to X-LC-HN in medium for 12 hours. Cell lysates were collected and immunoblot analysis was performed to examine the cleavage of SNARE proteins. As shown in FIG. 3B, X-LC-H _N entered neurons and cleaved VAMP2 in a concentration dependent manner. Lys-C activated X-LC-H _N showed significantly higher potency than intact X-LC-H _N : 10 nM activated X-LC-H _N cleaved similar levels of VAMP2 as 150 nM intact X-LC-H _N (Fig. 3B). It should be noted that intact X-LC-H _N is probably sensitive to proteolytic cleavage by cell surface proteases, so it is still active in neurons at high concentrations. Interestingly, activated X-LC-HN appears to be more potent than activated LC-HN BoNT/A (A-LC-HN) and BoNT/B (B-LC-HN), which did not show any or detectable cleavage of their SNARE substrates in neurons under the same assay conditions (FIG. 3B).

Peptide fragments of X-LC-HN under limited proteolysis were analyzed by TMT labeling quantitative mass spectrometry (Table 2).

His6-tagged recombinant X-LC-H _N was labeled with light TMT. Equal number of samples

- 66 041568

X-LC-HN was subjected to Lys-C and then labeled with heavy TMT. Then both samples were digested with chymotrypsin, combined together and subjected to quantitative analysis by mass spectrometry. A list of identified peptides is presented. The ratio of light TMT:heavy TMT is 2 times greater relative to each other for all peptides, except for five peptides (underlined), starting with N439. These five peptides showed no signal for labeling light TMT, indicating that N439 is a new N-terminus created by cutting Lys-C. Peptide sequences in table. 2 correspond, from top to bottom, SEQ ID NO: 94-226.

table 2

ScanF Theor. mass/charge PPM XCorr L Corr Ref. Peptide Start POSITION End POSITION Max. heavy Max. easy Heavy/light ratio 16633 2 611.384 1.46 1.751 0.651 xLcHn K.L]EINK#F.N 3 8 4.03E+04 2.92E+04 1.38 16638 2 606.3736 0.95 1.71 0.654 xLcHn K.LEINKF.N 3 8 4.03E+04 2.92E+04 1.38 15946 2 912.456 2.74 4.083 0.877 xLcHn F.N]YNDPIDGINV ITM*.R 9 22 4.34E+05 9.26E+05 0.47 15942 2 909.9508 2.28 4,546 0.661 xLcHn F.NYNDPIDGINVI TM*.R 9 22 4.34E+05 9.26E+05 0.47 17201 2 785.9092 1.26 2.455 0.273 xLcHn F.NYNDPIDGINVI. T 9 20 1.14E+06 1.94E+06 0.59 11082 2 679.833 1.17 1.74 0.754 xLcHn F.NYNDPIDGIN.V 9 18 8.01E+05 1.20E+06 0.67 11083 2 682.3382 0.96 2.6 0.742 xLcHn F.N]YNDPIDGIN.V 9 18 8.01E+05 1.20E+06 0.67 19628 2 535.3264 0.97 1.391 0.701 xLcHn D.P]IDGINVI.T 13 20 6.55E+04 1.23E+05 0.53 19626 2 532.8211 0.68 1.474 0.608 xLcHn D.PIDGINVI.T 13 20 6.55E+04 1.23E+05 0.53 20815 4 802.2073 2.19 2.962 0.582 xLcHn Y.N]PNYLNTPSE K#DEFLQGVIK#V L.E 78 99 4.57E+04 1.21E+05 0.38 20463 4 802.2073 1.96 2.726 0.385 xLcHn Y.N]PNYLNTPSE K#DEFLQGVIK#V L.E 78 99 2.96E+04 8.18E+04 0.36 20799 4 798.4495 1.91 3.647 0.659 xLcHn Y.NPNYLNTPSEK DEFLQGVIKVL.E 78 99 4.57E+04 1.21E+05 0.38 20568 4 798.4495 1.63 2.639 0.419 xLcHn Y.NPNYLNTPSEK DEFLQGVIKVL.E 78 99 2.96E+04 8.18E+04 0.36 22720 2 753.4631 1.96 2.339 0.222 xLcHn L.LELISSSIPLPL.V 112 123 1.40E+04 2.80E+04 0.50 21170 2 696.9211 1.9 1.781 0.326 xLcHn L.ELISSSIPLPL.V 113 123 2.75E+04 4.15E+04 0.66 21281 2 696.9211 1.86 2.099 0.282 xLcHn L.ELISSSIPLPL.V 113 123 2.75E+04 4.15E+04 0.66 21378 2 696.9211 1.83 1.593 0.149 xLcHn L.ELISSSIPLPL.V 113 123 2.75E+04 4.15E+04 0.66

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19246 2 578.363 1.18 1.443 0.27 xLcHn L.IJSSSIPLPL.V 115 123 l,61E+04 4.12E+04 0.39 19365 2 578.363 1.08 1.624 0.135 xLcHn L.IJSSSIPLPL.V 115 123 l,61E+04 4.12E+04 0.39 19241 2 575.8577 1 1.484 0.209 xLcHn L.ISSSIPLPL.V 115 123 l,61E+04 4.12E+04 0.39 19360 2 575.8577 0.91 1.673 0.298 xLcHn L.ISSSIPLPL.V 115 123 l,61E+04 4.12E+04 0.39 13952 2 948.9912 2.47 2.562 0.54 xLcHn L.V]SNGALTLSD NETIAY.Q 124 139 2.51E+05 3.67E+05 0.68 13949 2 946.486 2.42 1.729 0.599 xLcHn L.VSNGALTLSDN ETIAY.Q 124 139 2.51E+05 3.67E+05 0.68 6712 2 392.7318 0.19 1.523 0.206 xLcHn L.VSNGAL.T 124 129 l,18E+05 2.03E+05 0.58 10243 2 678.3482 0.73 1.499 0.616 xLcHn L.TJLSDNETIAY.Q 130 139 2.14E+05 3.67E+05 0.58 10242 2 675.843 0.69 1.594 0.842 xLcHn L.TLSDNETIAY.Q 130 139 2.14E+05 3.67E+05 0.58 15890 2 1110.579 2.74 2.243 θ,6 xLcHn L.QJANLVIYGPGP DIANNATY.G 150 168 5.20E+04 9.82E+04 0.53 15881 2 1108.073 1.86 2.121 0.673 xLcHn L.QANLVIYGPGP DIANNATY.G 150 168 5.20E+04 9.82E+04 0.53 11142 2 727.3879 1.39 1.945 0.635 xLcHn L.VIYGPGPDIAN N.A. 154 165 l,91E+04 3.90E+04 0.49 12879 2 894.962 1.24 2.673 0.731 xLcHn L.VIYGPGPDIAN NATY.G 154 168 6.62E+05 l,25E+06 0.53 10964 2 707.3541 2.8 1.39 0.466 xLcHn Y.GPGPDIANNAT Y.G 157 168 l,42E+04 2.24E+04 0.63 11091 2 456.2473 0.78 1.352 0.765 xLcHn N.ATYGLY.S 166 171 3.55E+04 8.08E+04 0.44 17435 2 1094.055 2.53 3.418 0.69 xLcHn Y.GJLYSTPISNGE GTLSEVSF.S 169 187 l,55E+05 2.27E+05 0.68 17410 2 1091.549 2.48 3.748 0.738 xLcHn Y.GLYSTPISNGE GTLSEVSF.S 169 187 l,55E+05 2.27E+05 0.68 19830 2 1259.631 2.15 2.885 0.676 xLcHn Y.GJLYSTPISNGE GTLSEVSFSPF.Y 169 190 l,98E+04 2.46E+04 0.81 20131 3 838.4197 2.08 2.781 0.742 xLcHn Y.GLYSTPISNGE GTLSEVSFSPF.Y 169 190 4.60E+05 5.32E+05 0.86 12546 2 862.9488 1.9 1.817 0.401 xLcHn Y.GJLYSTPISNGE GTLS.E 169 183 7.95E+04 7.83E+04 1.02 12571 2 860.4436 1.84 1.528 0.588 xLcHn Y.GLYSTPISNGE GTLS.E 169 183 7.95E+04 7.83E+04 1.02 19819 3 838.4197 1.61 2.655 0.662 xLcHn Y.GLYSTPISNGE GTLSEVSFSPF.Y 169 190 4.13E+04 4.59E+04 0.90 9948 2 851.4307 2.54 2.044 0.765 xLcHn Y.STPISNGEGTLS EVS.F 172 186 l,92E+04 2.39E+04 0.81 18409 2 1090.541 2.45 4.434 0.844 xLcHn Y.STPISNGEGTLS EVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 18417 2 1090.541 2.39 3.307 0.82 xLcHn Y.STPISNGEGTLS EVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 18418 2 1093.047 2.39 2.983 0.845 xLcHn Y.SJTPISNGEGTL SEVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 18876 2 1093.047 1.97 1,841 0.679 xLcHn Y.SJTPISNGEGTL SEVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 14591 2 927.4701 1.96 3.362 0.646 xLcHn Y.SJTPISNGEGTL SEVSF.S 172 187 2.09E+05 l,42E+05 1.48 14803 2 924.9649 1.88 1.934 0.462 xLcHn Y.STPISNGEGTLS 172 187 2.96E+05 4.65E+05 0.64

-68041568

EVSF.S 14852 2 924.9649 1.87 1.538 0.369 xLcHn Y.STPISNGEGTLS EVSF.S 172 187 2.96E+05 4.65E+05 0.64 18554 2 1093.047 1.72 3.044 0.86 xLcHn Y.S]TPISNGEGTL SEVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 18552 2 1090.541 1.68 3.643 0.813 xLcHn Y.STPISNGEGTLS EVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 14978 2 927.4701 1.68 1.401 0.31 xLcHn Y.S]TPISNGEGTL SEVSF.S 172 187 4.96E+04 7.37E+04 0.67 18680 2 1093.047 1.66 1.656 0.717 xLcHn Y.S]TPISNGEGTL SEVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 20660 2 1093.047 1.63 2.182 0.831 xLcHn Y.S]TPISNGEGTL SEVSFSPF.Y 172 190 5.24E+05 5.44E+05 0.96 17226 2 924.9649 1.62 2.459 0.698 xLcHn Y.STPISNGEGTLS EVSF.S 172 187 3.25E+05 5.31E+05 0.61 17306 2 924.9649 1.62 2.126 0.588 xLcHn Y.STPISNGEGTLS EVSF.S 172 187 3.25E+05 5.31E+05 0.61 10139 2 696.3644 1.61 1.942 0.628 xLcHn Y.S]TPISNGEGTL S.E. 172 183 6.39E+04 8.46E+04 0.76 18674 2 1090.541 1.59 2.423 0.765 xLcHn Y.STPISNGEGTLS EVSFSPF.Y 172 190 8.21E+05 9.22E+05 0.89 20642 2 1090.541 1.55 2.608 0.823 xLcHn Y.STPISNGEGTLS EVSFSPF.Y 172 190 5.24E+05 5.44E+05 0.96 8077 2 696.3644 1.54 1.763 0.707 xLcHn Y.S]TPISNGEGTL S.E. 172 183 l,09E+05 l,29E+05 0.84 18476 2 562.2872 1.17 1.812 0.784 xLcHn L. SEVSFSPF.Y 183 190 4.49E+04 7D1E+04 0.63 18478 2 564.7924 0.95 1.57 0.965 xLcHn L.S]EVSFSPF.Y 183 190 4.49E+04 7D1E+04 0.63 13190 3 652.0048 0.97 2.099 0.697 xLcHn F.Y]LK#PEDESYG NY.R 191 202 l,98E+06 3.09E+06 0.64 12839 3 540.6289 0.38 2.441 0.423 xLcHn F.Y]LK#PEDESY.G 191 199 2.43E+05 5.45E+05 0.44 7115 2 538.7403 0.69 1.476 0.615 xLcHn F.D]ESYGNY.R 196 202 l,44E+05 ZD4E+05 0.46 16124 3 661.3911 1.14 2.123 0.575 xLcHn Y.G]NYRSLVNIV NK#F.V 200 212 2.81E+04 2.27E+04 1.24 11646 3 474.5894 0.49 2.077 0.602 xLcHn H.NLYGISNRNF.Y 235 244 2.80E+05 4.38E+05 0.64 10091 4 603.8103 1.23 2.98 0.756 xLcHn F.YYNFDTGKIET SRQQN.S 245 260 3.05E+05 l,94E+05 1.57 9932 4 603.8103 1.08 2.5 0.638 xLcHn F.YYNFDTGKIET SRQQN.S 245 260 3.05E+05 l,94E+05 1.57 10782 3 820.4359 0.65 2.156 0.438 xLcHn Y.Y]NFDTGK#IET SRQQNSL.I 246 262 7.40E+04 l,33E+05 0.56 15039 3 819.1317 1.41 2.281 0.516 xLcHn L.ISERLNTVTVEN DLLKY.I 298 314 l,45E+05 2.76E+05 0.53 13824 3 619.6838 1.27 2.021 0.44 xLcHn L.NTVTVENDLLK Y.I 303 314 ZD0E+04 5.07E+04 0.61 10975 2 760.4251 1.45 2.089 0.663 xLcHn F.V]LNESNLAQRF ,S 345 355 4.63E+05 6.60E+05 0.70 17696 3 966.86 2.14 2.704 0.532 xLcHn H.Y]LK#ERPIDPIY VNILDDNSY.S 363 382 8.61E+04 l,42E+05 0.61 20823 2 827.9299 2.21 2.495 0.806 xLcHn D.P]IYVNILDDNS Y.S 371 382 7.80E+04 8D6E+04 0.96

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20825 2 825.4247 2.17 2.958 0.881 xLcHn D.PIYVNILDDNS Y.S 371 382 7.80E+04 8.16E+04 0.96 12614 2 685.3561 1.34 1.843 0.609 xLcHn N.I]LDDNSYSTL.E 376 385 l,01E+05 2.57E+05 0.39 11272 2 441.7421 1.48 1.795 0.903 xLcHn Y.S]TLEGF.N 383 388 4.45E+04 l,36E+05 0.33 11261 2 439.2369 1.08 1.536 0.514 xLcHn Y.STLEGF.N 383 388 4.45E+04 l,36E+05 0.33 14260 2 441.7421 0.49 1.462 0.848 xLcHn Y.S]TLEGF.N 383 388 5.41E+04 l,32E+05 0.41 14246 2 439.2369 0.38 1.46 0.844 xLcHn Y.STLEGF.N 383 388 5.41E+04 l,32E+05 0.41 13808 2 916.4629 2.48 2.036 0.498 xLcHn F.NISSQGSNDFQ GQLL.E 389 403 2.86E+04 6.39E+04 0.45 17314 3 815.7238 2.05 2.149 0.621 xLcHn F.NISSQGSNDFQ GQLESSYF.E 389 408 l,26E+05 2.71E+05 0.47 6340 2 803.3788 0.75 2.554 0.721 xLcHn F.NISSQGSNDFQ GQ.L 389 401 l,03E+04 2.21E+04 0.47 11696 2 493.2814 0.67 1.62 0.518 xLcHn L.L]YNAIY.R 429 434 l,19E+05 l,87E+05 0.63 11692 2 490.7762 θ,6 1.57 0.118 xLcHn L.LYNAIY.R 429 434 l,19E+05 l,87E+05 0.63 14021 3 756.4332 2.14 2.305 0.363 xLcHn K.N1YLNNIDLED K#K#T,T 439 451 6.85E+05 5.12E+03 133 95 12997 3 1009.909 1.93 3.044 0.553 xLcHn K.N1YLNNIDLED K#K#TTSK#TN,V 439 456 4.24E+05 0.00E+00 #DI V/0! 12932 3 1009.909 1.92 4.251 0.695 xLcHn K.N1YLNNIDLED K#K#TTSK#TN,V 439 456 4.24E+05 0.00E+00 #DI V/0! 14003 3 790.1157 1.59 2,894 0.542 xLcHn K.N1YLNNIDLED K#K#TT.S 439 452 4.40E+06 L13E+04 389, 24 13105 4 729.173 1 3 0.599 xLcHn K.N1YLNNIDLED K#K#TTSK#T,N 439 455 3.39E+06 3.14E+03 1078 .97 11567 3 747.091 1.33 2.711 0.518 xLcHn N.YLNNIDLEDKK TT.S 440 452 l,22E+04 9.52E+04 0.13 11515 3 747.091 1.33 2.729 0.496 xLcHn N.YLNNIDLEDKK TT.S 440 452 l,22E+04 9.52E+04 0.13 17001 2 857.9579 1.99 1.967 0.713 xLcHn G.CIEVENKDLF.L 467 476 3.29E+04 l,64E+05 0.20 16337 4 941.793 1.76 2.419 0.135 xLcHn F.L]ISNK#DSLNDI NLSEEK#IK#PETT VF.F 477 501 l,27E+06 l,06E+06 1.19 16394 4 941.793 1.72 2.19 0.391 xLcHn F.L]ISNK#DSLNDI NLSEEK#IK#PETT VF.F 477 501 l,27E+06 l,06E+06 1.19 17983 3 993.888 2.4 3.193 0.657 xLcHn K.D]SLNDINLSEE K#IK#PETTVF.F 482 501 3.27E+04 2.59E+03 12.6 4 18031 3 955.5456 2.06 3.607 0.764 xLcHn D.S]LNDINLSEEK #IK#PETTVF.F 483 501 4.90E+04 4.12E+04 1.19 11720 2 834.8912 2.14 1.689 0.726 xLcHn L.SNYDFTE ANSIP S.I 514 526 2.91E+04 2.50E+04 1.17 13409 2 1065.513 2.12 4.742 0.769 xLcHn L.S]NYDFTEANSI PSISQQ.N 514 530 7.05E+04 7.52E+04 0.94 8896 2 642.7908 1.42 1.851 0.708 xLcHn L.SNYDFTE AN.S 514 522 2.12E+05 2.27E+05 0.94 8883 2 645.296 1.39 2.362 0.879 xLcHn L.S]NYDFTEAN.S 514 522 2.12E+05 2.27E+05 0.94 17774 2 1051.044 2.7 1.967 0.9 xLcHn Y.DFTEANSIPSIS QQNIL.E 517 533 3.52E+04 3.28E+04 1.07

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17438 2 1051.044 2.55 1.944 0.665 xLcHn Y.DFTEANSIPSIS QQNIL.E 517 533 9.04E+04 8.08E+04 1.12 12784 2 880.9387 1.87 2.537 0.778 xLcHn Y.DFTEANSIPSIS QQN 517 530 4.72E+05 5.06E+05 0.93 12779 2 883.4439 1.73 3.423 0.797 xLcHn Y.D]FTEANSIPSIS QQN 517 530 4.72E+05 5.06E+05 0.93 16126 4 911.7114 1.6 3.934 0.579 xLcHn Y.DFTEANSIPSIS QQNILERNEELYE PIRN.S 517 545 5.41E+05 2.02E+05 2.67 12449 2 937.9602 1.56 3.012 0.73 xLcHn Y.DFTEANSIPSIS QQN.I 517 531 5.16E+05 5.72E+05 0.90 12151 2 940.4654 1.47 1.476 0.458 xLcHn Y.D]FTEANSIPSIS QQN.I 517 531 4.65E+05 4.96E+05 0.94 20080 3 1106.923 2.52 3.592 0.677 xLcHn N.S]IPSISQQNILE RNEELYEPIRNSL F.E. 523 548 l,86E+04 l,52E+04 1.23 11314 3 700.7002 1.59 2.563 0.763 xLcHn L.TDSVDEALSNP NKVY.S 583 597 l,74E+05 l,84E+05 0.95 11315 3 704.0405 1.56 2.444 0.262 xLcHn L.T]DSVDEALSNP NK#VY.S 583 597 l,74E+05 l,84E+05 0.95 11916 3 700.7002 1.52 2.049 0.508 xLcHn L.TDSVDEALSNP NKVY.S 583 597 2.27E+05 2.78E+05 0.82 7902 2 635.3637 0.25 1.478 0.567 xLcHn L.SNPNKVY.S 591 597 6.79E+05 8.90E+05 0.76 7903 2 640.3742 0.25 1.485 0.282 xLcHn L.S]NPNK#VY.S 591 597 6.79E+05 8.90E+05 0.76 14536 3 927.1457 1.59 2.027 0.553 xLcHn Y.S]PFK#NM*SNT INSIETGITSTY.I 598 618 l,47E+05 8.07E+04 1.83 14487 3 923.8054 1.54 2.35 0.708 xLcHn Y.SPFKNM*SNTI NSIETGITSTY.I 598 618 l,47E+05 8.07E+04 1.83 13108 3 813.421 2.03 2.046 0.548 xLcHn F.KNM*SNTINSIE TGITSTY.I 601 618 2.41E+06 2.42E+06 1.00 12962 3 813.421 1.98 2.378 0.482 xLcHn F.KNM*SNTINSIE TGITSTY.I 601 618 2.41E+06 2.42E+06 1.00 13079 3 813.421 1.98 2.245 0.617 xLcHn F.KNM*SNTINSIE TGITSTY.I 601 618 2.41E+06 2.42E+06 1.00 13194 2 915.4701 2.12 1.504 0.654 xLcHn M.S]NTINSIETGIT STY.I 604 618 l,74E+05 3.22E+05 0.54 13170 2 912.9649 1.99 1.791 0.699 xLcHn M.SNTINSIETGIT STY.I 604 618 l,74E+05 3.22E+05 0.54 12405 2 812.4274 2.38 1.349 0.781 xLcHn N.TINSIETTGITSTY .1 606 618 2.94E+04 3.41E+04 0.86 14318 3 837.465 1.53 2.439 0.647 xLcHn F.S]DETGK#IDVI DK#SSDTL.A 632 648 2.23E+04 l,55E+04 1.44 19535 2 586.368 0.52 1.753 0.731 xLcHn L.A]IVPYIGPL.L 649 657 7.93E+05 l,16E+06 0.69 19564 2 442.268 0.43 1.498 0.892 xLcHn V.PYIGPL.L 652 657 l,30E+04 2.64E+04 0.49 9998 2 715.396 1.76 2.148 0.33 xLcHn V.I]GGELAREQVE .A 699 709 8.53E+04 l,32E+04 6.44 5372 3 494.9536 0.34 2.943 0.626 xLcHn L.SRQANAIKM*.N 754 762 2.50E+05 l,57E+04 15.9 7 5363 3 494.9536 0.32 3.357 0.734 xLcHn L.SRQANAIKM*.N 754 762 2.50E+05 l,57E+04 15.9 7 17794 2 734.3537 1.89 2.212 0.79 xLcHn F.SEEDDLINQY.K 879 888 3.00E+05 4.05E+05 0.74

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14171 4 757.641 0.97 2.289 0.421 xLcHn F.DDLINQYKNEG SILPETGGLEHH.H 882 904 l,02E+04 3.96E+04 0.26 7676 4 679.3532 1.49 2.624 0.624 xLcHn Y.KNEGSILPETG GLEHHHHHH.- 889 908 3.63E+04 3.74E+04 0.97 13193 3 588.3365 1.18 2.307 0.629 xLcHn Y.KNEGSILPETG GL.E 889 901 2.81E+05 3.08E+05 0.91 10362 3 677.037 1.15 2.582 0.615 xLcHn Y.KNEGSILPETG GLEH.H 889 903 8.01E+05 1D7E+06 0.68 9325 4 542.2943 1.06 2.617 0.58 xLcHn Y.KNEGSILPETG GLEHH.H 889 904 2D7E+05 2.96E+05 0.73

The unique feature of the disulfide bond in BoNT/X.

The linker site of BoNT/X contains an additional cysteine (C461) that is unique to BoNT/X. To determine which cysteine forms the disulfide bond connecting LC and HC, three X-LC-H _N mutants were created with each of the three mutant cysteine residues (C423S, C461S and C467S). These three cysteine mutants, as well as wild-type (WT) X-LC-H _N , were subjected to limited proteolysis and then analyzed by SDS-PAGE and Coomassie blue staining with or without DTT reducing agent (FIG. 3C). A mutated cysteine in LC (C423S) was found to result in a protein that split into two bands of ~50 kDa with or without DTT, indicating that C423S abrogated the interchain disulfide bond. In contrast, mutants containing either C461S or C467S showed a single band at 100 kDa in the absence of DTT, which split into two ~50 kDa bands in the presence of DTT, suggesting that both C461 and C467 in _HN can form an interchain disulfide bond with C423 in LC. Also, the X-LC-H _N (C423S) mutant appears to be more susceptible to Lys-C than both C461S and C467S mutants, resulting in further protein cleavage (FIG. 3C). This result suggests that the loss of the interchain disulfide bond can increase the freedom of LC and H _N , thereby exposing more surface areas. In addition, a portion of the X-LC-H _N WT formed aggregates at the top of the SDS-PAGE gel (FIG. 3C). These aggregates are due to the formation of an intermolecular disulfide bond, since they disappeared in the presence of DTT (Fig. 3C, + DTT). C423, C461 and C467 are the only three cysteines in X-LC-H _n . Mutation of any of the three cysteines destroyed the X-LC-H _N aggregates (FIG. 2C, - DTT), indicating that the formation of an intermolecular disulfide bond is due to the presence of an additional cysteine in the linker site.

Most of the activated X-LC-H _N WT also separated into two ~50 kDa bands on an SDS-PAGE gel without DTT (FIG. 3C). On the other hand, X-LC-H _N WT is as resistant to Lys-C as the C461S and C467S mutants and did not show further degradation as did the C423S mutant (Fig. 3C, + DTT), confirming that X-LC-H _N WT differs from the C423S mutant. One possible explanation is disulfide shuffling due to the presence of two cysteines close to each in H _n (C461 and C467), which can rearrange the disulfide bond from interchain C423-C467 or C423-C467 to intrachain C461-C467 under denatured conditions ^{48 .49} . To test this hypothesis, an alkylating reagent, N-ethylmaleimide (NEM), was used, which reacts with free cysteine sulfhydryls and permanently blocks any free cysteines. As shown in FIG. 3D, X-LC-H _n WT pretreated with NEM showed mostly one band at 100 kDa in the absence of DTT and split into two bands of ~50 kDa in the presence of DTT. These results confirmed that native X-LC-H _N WT contains mainly an interchain disulfide bond that is susceptible to disulfide shuffling due to the presence of a third cysteine at the linker site.

Finally, the activity of three X-LC-H _N cysteine mutants in cultured neurons was examined. As shown in FIG. 3E, mutating cysteine in LC (C423S) abolished X-LC- _HN activity, as evidenced by the absence of VAMP2 cleavage in neurons. Mutation of one of the two cysteines in H _N (C461 or C467) did not significantly affect the activity of X-LC-H _N compared to wild-type (WT) X-LC-H _n (FIG. 3E). These results confirmed that the interchain disulfide bond is important for BoNT/X activity and demonstrated that a functional interchain disulfide bond can be formed through either C423-C461 or C423-C467.

Creation of full length BoNT/X by sortase mediated ligation.

To evaluate whether BoNT/X is a functional toxin, it was necessary to create and test full length BoNT/X. However, BoNTs are among the most dangerous potential bioterrorist weapons. Therefore, the necessary precautions were taken and a full length active toxin gene was not generated. Instead, an approach was developed to generate limited amounts of full length BoNT in test tubes under controlled conditions by enzymatic ligation of two non-toxic BoNT fragments. This method uses a transpeptidase known as sortase, which recognizes specific peptide motifs and covalently links two peptides together to form a native peptide bond (FIG. 4A). This approach has previously been used to design chimeric toxins and other fusion proteins ^50,51 .

-72041568

An engineered sortase A, known as SrtA*, was created from Staphylococcus aureus ⁵¹ . SrtA* recognizes the LPXTG (SEQ ID NO: 57) peptide motif, cleaves between TGs, and simultaneously forms a new peptide bond between the protein containing LPXTG (SEQ ID NO: 57) with other proteins/peptides containing one or more N-terminal glycines (Fig. 4A). Two non-toxic BoNT/X fragments were generated: (1) LC-H _N with LPETGG motif (SEQ ID NO: 58) and C-terminally fused His6 tag; (2) H _C BoNT/X (X-HC) with a GST tag and a thrombin cleavage site at its N-terminus. Cutting with thrombin releases X-H _with free glycine at the N-terminus. Incubation of these two fragments with SrtA* generated a limited amount of full length ~150 kDa BoNT/X containing a short linker (LPETGS, SEQ ID NO: 59) between LC-H _N and _HC (FIGS. 4A, 4B).

It was observed that X-HC showed a strong tendency to aggregate in solution for unknown reasons when cut from the GST label, which may be the reason for the low ligation efficiency for BoNT/X (FIG. 4B). In contrast, ligation of X-LC-HN to H _C BoNT/A (A-HC) using the same approach achieved much higher efficiency, with most X-LC-Hn ligated into the full length chimeric CA toxin (Fig. 8A). .

BoNT/X is active in cultured neurons.

To analyze full length BoNT/X activity, cultured rat cortical neurons were used as a model system. Neurons were subjected to mixtures for sortase-mediated ligation and various control mixtures in the environment. Cell lysates were harvested 12 hours later and immunoblot analysis was performed to check for cleavage of SNARE proteins. As shown in FIG. 4C, X-LC-HN separately cleaved some VAMP2 due to its high concentration in the reaction mixture. The control mixture containing X-LC-HN and X-HC rather than sortase slightly increased VAMP2 cleavage compared to X-LC-HN alone. This result confirms that X-HC can be linked to X-LC-HN by non-covalent interactions. This interaction appears to be specific as the control mixture containing X-LC-HN and A-Hc showed the same level of VAMP2 cleavage as X-LC-HN alone (FIG. 8B). Ligation of X-LC-HN and X-HC with sortase enhanced VAMP2 cleavage at a mixture of X-LC-HN and X-HC without sortase (Fig. 4C), demonstrating that full-length ligated BoNT/X can enter neurons and cleave VAMP2. Similarly, a full length ligated chimeric CA toxin also entered neurons and cleaved VAMP2 (FIG. 8B).

Mixing X-HC with X-LC-HN increased the number of aggregates at the top of the SDS-PAGE gel compared to X-LC-HN alone. These aggregates disappeared in the presence of DTT, suggesting that a portion of X-HC formed an intermolecular disulfide bond with X-LC-HN. The presence of DTT also increased the amount of full length ligated BoNT/X, indicating that a portion of BoNT/X had aggregated via an intermolecular disulfide bond (FIG. 4B). The formation of these aggregates can significantly reduce the effective concentrations of the toxin monomer in solution. This may be an inherent shortcoming of the BoNT/X sequence. X-HC contains one cysteine (C1240) and mutating this cysteine did not affect the activity of ligated BoNT/X (FIG. 9). In addition, the C1240S mutant can be combined with the C461S or C467S mutations in X-LC-HN to create a modified BoNT/X without free cysteines (FIG. 9). These mutant toxins maintained the same activity levels as BoNT/X WT, but were more stable in solution as monomers than BoNT/X WT.

BoNT/X Induced Atrophic Paralysis in Mice In Vivo BoNT/X activity was tested in vivo using a well-established non-lethal assay in mice known as finger abduction score (DAS). This assay measures localized muscle paralysis following injection of BoNT into mouse hindlimb muscles ^{52, 53} . BoNT causes atrophic paralysis of the muscles of the limb, which can be identified by the inability to spread the fingers during the startle reflex. The sortase-activated reaction mixture (FIG. 4B, lane 7) was injected into the gastrocnemius muscles of the right hind limb in mice. Within 12 hours, typical atrophic paralysis developed in the right limb, and the fingers were unable to splay (Fig. 4D). These results confirm that BoNT/X is capable of inducing atrophic paralysis in vivo like other BoNTs.

BoNT/X was not recognized by antisera raised against all known BoNTs.

To further confirm that BoNT/X is a serologically unique BoNT, dot blot analyzes were performed using antisera raised against known BoNTs, including all seven serotypes, as well as one mosaic toxin (BoNT/DC). Four horse antisera (trivalent anti-BoNT/A, B and E, anti-BoNT/C, anti-BoNT/DC and anti-BoNT/F) and two goat antisera (anti-BoNT/G and anti-BoNT) were used. /D). All of these antisera were able to neutralize their respective target BoNTs and prevent the cleavage of SNARE proteins in neurons (FIG. 10), thereby confirming their specificity and efficacy. As shown in FIG. 4E, these antisera recognized their respective target toxins, but none of them recognized BoNT/X. It should be noted that the antisera raised against BoNT/DC and BoNT/C cross-reacted with each other because they have a high degree of similarity in their H _C . This result proved that BoNT/X is a new serotype of BoNT.

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Inactive BoNT/X full length.

Finally, the possibility of obtaining BoNT/X as a soluble protein was investigated. To meet the biosafety requirement, mutations were introduced into LC BoNT/X that inactivate its toxicity. Mutations at two residues R362A/Y365F in BoNT/A have been shown to inactivate LC protease activity in vitro and abolish full-length BoNT/A toxicity in mice in vivo ^54-56 . These two residues are conserved in all BoNTs, including BoNT/X. Therefore, appropriate mutations were introduced at these two sites (R360A/Y363F in BoNT/X). As shown in FIG. 4F, this full-length inactivated form of BoNT/X (BoNT/XRY) was generated and purified as a His6-tagged protein in E. coli recombinantly. It does not have any neuronal activity since VAMP2 is not cleaved in neurons (FIG. 11).

A significant portion of BoNT/XRY formed aggregates at the top of the SDS-PAGE gel (FIG. 4F). This is likely due to the formation of an intermolecular disulfide bond from additional cysteine in the linker site and cysteine in _HC , since the addition of DTT converted the aggregates to monomeric BoNT/X _RY (Fig. 4F). Mutation of these cysteines does not affect BoNT/X activity (FIG. 9) and is beneficial in preventing intermolecular disulfide bond formation and BoNT/X aggregation.

The inactive form of BoNT/X can be used as a carrier medium for delivery of therapeutic agents to neurons. Inactivation can be achieved by mutations at any of the following residues or combinations thereof: R360, Y363, H227, E228, or H231, with the last three residues forming a conserved protease motif.

Purification of inactive full length BoNT/X on an industrial scale.

The possibility of purifying full length BoNT/X to a high purity and in good yield was investigated, which would be important for the industrial production of BoNT/X (or its derivatives) as a therapeutic toxin. Several cell growth and expression parameters were tested, such as temperature, induction time, and IPTG concentrations. The optimal parameters chosen for protein expression were culturing cells at 37°C until they reached exponential growth, at this stage the temperature was reduced to 18°C, and expression was induced by adding 1 mM IPTG to the medium. The cells were then cultured for 16-18 h prior to harvest. The presence of BoNT/X was confirmed by SDS-PAGE and showed a high level of overexpression in the soluble fraction (FIG. 11B).

Several laboratory scale purification tests were performed to optimize the preparation process. Mechanical cell lysis using Emulsiflex-C3 (Avestin, Mannheim, Germany) was the preferred method for intracellular protein extraction and proved to be more efficient than sonication. Various buffer conditions were also to be evaluated for optimal recovery of BoNT/X. The reducing agent was included in the cleaning process and significantly reduced the tendency to undesirable aggregation. In addition, glycerol was used as an additive at an early stage of the purification process and improved protein stability.

The BoNT/X construct was expressed with a His6 tag that can be used for affinity chromatography as a first purification step. For laboratory scale testing, a 5 ml HIsTrapFF column (GE Healthcare, Danderyd, Sweden) was used. Various concentrations of imidazole were tested to achieve maximum purity of the original chromatography. BoNT/X was eluted from 100 mM imidazole; however, the main impurity was easily cleared together with the toxin. This impurity appears to interact non-specifically with BoNT/X and was identified by mass spectrometry as an E. coli host protein (arnA bifunctional polymyxin resistance protein). The presence of this impurity was drastically reduced by introducing a high salt concentration (500 mM NaCl) and by carrying out an additional washing step at 100 mM imidazole during purification. This allowed a purer BoNT/X fraction to be eluted at 250 mM imidazole. This later fraction can then be processed by size exclusion chromatography.

This protocol was then scaled up to 12 liters of medium under the conditions described above. In addition, a higher affinity chromatography matrix was prepared, consisting of 15 ml of Protino® Ni-NTA agarose (Macherey-Nagel, Duren, Germany) to increase the yield of recovered BoNT/X. The final purification step was performed by size exclusion chromatography using a Superdex200-16/60 column (GE Healthcare, Danderyd, Sweden). Purities of 85 to 90% were achieved using this method (FIG. 11C). The protein could be concentrated (using a Vivaspin concentrator with a 100 kDa threshold; GE Healthcare, Danderyd, Sweden) and seemed stable up to 10 mg/mL. Full details of the acquisition process are described below. The output of the resulting BoNT/X was approximately 3 mg per 1 liter of cell culture. Together, these results demonstrated that BoNT/X can be purified industrially to a high degree of purity.

It should be noted that the purification was performed in the presence of a reducing agent that could reduce the disulfide bond between LC and HC so that the purified toxin could not be active. However, the engineered BoNT/X derivative containing mutations at the cysteine sites (one mutation per

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C461 or C467 combined with a mutation in C1240) could be purified without reducing agents. It should be noted that the inactive form of BoNT/X (and its derivative with cysteine mutations) can be used as a carrier medium for the delivery of therapeutic agents to neurons. Inactivation can be achieved by mutations at any of the following residues or combinations thereof: R360, Y363,

H227, E228 or H231 (last three residues from the conserved protease motif).

Identification of gangliosides as receptors for BoNT/X.

Gangliosides are well-established lipid co-receptors for all BoNTs, and the ganglioside binding motif is quite conserved in BoNT/X (FIG. 1C). Highly purified inactive full length BoNT/X was used to study the binding of BoNT/X to neuronal cells via gangliosides. An in vitro ELISA assay was developed to test interactions with the four major brain gangliosides: GD1a, GD1b, GT1b, and GM1. A-LC was used as a negative control to evaluate non-specific binding. A direct comparison was made with the BoNT/A receptor binding domain (A-HC). Protein binding was detected using an anti-His6 tag antibody. BoNT/X was found to exhibit dose-dependent binding to all four gangliosides at the level of non-specific A-LC binding (FIG. 12), confirming that BoNT/X is able to use all four brain gangliosides as co-receptors. Consistent with previous reports, BoNT/A gave equal preference to GD1a and GT1b (FIG. 12F) and their terminal fragment NAcGal-Gal-NAcNeu (with apparent EC ₅₀ values of 0.7 and 1.0 μM, respectively, using a sigmoidal dose-response model) . In contrast, BoNT/X showed higher affinity for GD1b and GM1 compared to GD1a and GT1b (FIG. 12E). This suggests that BoNT/X has a preferred sialic acid recognition pattern, also seen in BoNT/B and TeNT. BoNT/X has a conserved SxWY motif in a homologous location with one of the other toxins. The fact that it can recognize all four gangliosides, albeit with low affinity, may be indicative of multiple carbohydrate binding sites.

Discussion.

Eight BoNT serotypes have been identified in the 45 years since the identification of the last major BoNT serotype. BoNT/X has the least protein sequence identity to any other BoNT and TeNT from this toxin family, and this low level of identity is evenly distributed throughout the sequence of the toxin. As expected, BoNT/X was not recognized by any antisera raised against known BoNTs. It clearly represents a unique and distinct evolutionary branch of the toxin family.

BoNT/X was identified by searching the genomic sequences of strains of Clostridium botulinum and represents the first major type of toxin identified using genome sequencing and a bioinformatics approach. Strain 111, which contains the BoNT/X gene, was originally identified in an infant patient with botulism in the 1990s. Preliminary characterization using a classical neutralization assay determined that BoNT/B2 is the main toxin of this strain. It is likely that BoNT/X is a silent gene for the toxin or that it was not expressed at detectable levels of toxicity under culture conditions in the laboratory. Therefore, it can only be identified by sequencing strain 111. This illustrates the importance of genomic sequencing and bioinformatics approaches to understanding microbial virulence factors.

Silent BoNT genes have often been identified in various strains of Clostridium botulinum. It is not clear why these bacteria contain the silent toxin gene. It may be an evolutionarily degenerate gene. This is clearly the case when the silent toxin genes contain premature stop code mutations. However, there are also cases where the silent gene encodes a full length BoNT. Whether these full-length silent BoNTs can be expressed and exhibit toxicity under certain environmental conditions remains an intriguing question.

The general three-domain structures and functions of BoNT are well preserved in BoNT/X, but it also has a few unique characteristics: (1) it uses VAMP as its target in neurons, just like BoNT/B, D, F, and G, but it cuts VAMP at a new site (R66-A67 in VAMP2) that is unique to this toxin. This further expands the repertoire of toxins that can be used to remove VAMP at different sites. (2) The interchain disulfide bond connecting LC and H _N is retained in BoNT/X, but it also contains a unique additional cysteine at the linker site, which can lead to disulfide shuffling. The additional cysteine on H _N is not essential for LC-H _N activity (FIG. 3D) and mutating it has the advantage of preventing intermolecular disulfide bond formation (FIG. 3D, 4B).

The His6-tagged X-LC-H _N fragment is stable in buffers as recombinant proteins. It showed a higher level of activity on neurons than A-LC-H _N and B-LC-H _N (Fig. 3B), suggesting that its membrane translocation and/or protease activity may be more efficient than that of the respective fragments. in BoNT/A and BoNT/B. X-LC-H _N may be a useful reagent for targeting VAMP1/2/3 in a wide range of cell types and tissues because its penetration

- 75 041568 may not be limited to neurons. For example, it could potentially be used to reduce pain in a local area by targeting both sensory neurons and other cells that secrete inflammatory signals. It can also be used to produce chimeric toxins such as Xa (FIG. 8).

X-HC is functional as its presence enhances VAMP2 cleavage in neurons compared to LC-HN alone (Fig. 4C). However, XH _C may have some unfavorable characteristics that have yet to be assessed. For example, sufficient levels of soluble XH _C were obtained only when it was fused with GST, which is known to facilitate protein folding/solubility, but not with the His6 tag. After being freed from the GST tag, X-HC tends to aggregate. In addition, cysteine in X-HC can also form an intermolecular disulfide bond (FIG. 4B). Inactive full length BoNT/X can be purified and can exist as a soluble protein, suggesting that the solubility problem with X-HC may be at least in part due to separation of this domain from X-LC-HN. For example, X-LC-HN can interact with X-HC and cover its potential hydrophobic segments in a full length context, which is not unusual for a multidomain protein.

Gangliosides have long been established as neuronal receptors for all BoNT subtypes. It has been shown that BoNT/X can bind to all four of the most common gangliosides: GD1a, GD1b, GT1b and GM1. In addition, it is characterized by a marked difference in affinity and specificity compared to BoNT/A. This is an intriguing property, as other BoNTs appear to have varying degrees of preference for the ganglioside subgroup. For example, BoNT/A, E, F and G prefer GD1a and GT1b. BoNT/X can potentially recognize a wider range of neuron types than other BoNTs.

It is possible that BoNT/X has low in vivo toxicity, which may explain why BoNT/X activity was not detected in the original 111 strain study. If so, the reduced toxicity is likely due to its HC domain, since X- LC-HN appears to be more active than A-LC-H _n and B-LC-H _N . The formation of an intermolecular disulfide bond can also reduce the effective concentration of the toxin. To determine its activity in vivo, full length native BoNT/X will need to be generated, but it will be important to generate a neutralizing antiserum using non-toxic BoNT/X fragments prior to full length toxin production.

The introduction of a full length active toxin gene into any expression systems/organisms is always a major biosecurity concern and has become a major hurdle in the study of the structural functions of biological toxins. This is especially important for BoNT, as they are one of six potential Category A bioterrorist weapons ⁴ . In accordance with the present invention, a method was developed for the biochemical assembly of a limited amount of a full length toxin from two complementary and non-toxic fragments. Each fragment is expressed and purified individually and then ligated together with sortase in test tubes. Other methods of protein ligation, such as intein cleavage systems, which fuse two protein fragments via protein trans-splicing, can also be used ⁵⁷ . By controlling the amount of precursor fragments in the reaction, the amount of full-length ligated toxin can be tightly controlled. This semi-synthetic approach can be used to produce multi-domain biological toxins and other toxic proteins under controlled conditions. It also provides a versatile platform for creating fusion and chimeric toxins, such as substituting HCs for two BoNTs, replacing HC BoNTs with other target proteins, or attaching additional cargo to toxins. Since a full-length cDNA toxin is never formed, and there is no expression of toxins in bacteria or any other living organism, this approach greatly mitigates the biosecurity concerns associated with the production of wild-type and mutant toxins and will greatly facilitate studies of the structural functions of biological toxins and toxic proteins. .

Materials and methods/

Materials: Mouse monoclonal antibodies against syntaxin 1 (HPC-1), SNAP-25 (C171.2) and VAMP2 (C169.1) were kindly provided by E. Chapman (Madison, WI) and available from Synaptic Systems (Goettingen, Germany) . Mouse anti-actin monoclonal antibody was purchased from Sigma (AC-15). Equine polyclonal antiserum against BoNT/A/B/E, BoNT/C, BoNT/DC, BoNT/F and goat polyclonal antiserum against BoNT/G were obtained from the FDA. Goat anti-BoNT/D polyclonal antibody was purchased from Fisher Scientific (NB10062469). BoNT/A, BoNT/B, BoNT/C, BoNT/DC, BoNT/E, BoNT/F and BoNT/G were purchased from Metabiologics (Madison, WI). BoNT/D was kindly provided by E. Johnson (Madison, WI).

cDNA and constructs: Synthesized cDNA encoding X-LC (residues 1-439) and XH _C (residues 893-1306). The cDNA encoding XH _N was generated in-house using the Gibson assembly method. cDNA encoding A-LC (residues 1-425, M30196) and B-LC (residues 1-439, AB232927) were synthesized using GenScript (New Brunswick, NJ). These LCs were cloned into pET28 vectors for expression as His6-tagged proteins. XH _C was cloned into pGEX4T for expression as

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GST-tagged protein. X-LC-HN, A-LC-HN and B-LC-HN were subcloned into the pET28 vector, with the LPETGG peptide sequence (SEQ ID NO: 58) fused to their C-terminus, and purified as His6-tagged proteins. The inactive full length form of BoNT/X was assembled in-house from mutant X-LC (R360A/Y363F), X-HN and XH _c . It was cloned into the pET28 vector, with the His6 tag fused to the C-terminus of BoNT/X. The cDNA encoding rat VAMP2 was kindly provided by E. Chapman (Madison, WI). VAMP2 (1-96) was cloned into the pET28 vector and expressed as a His6-tagged protein. VAMP2 (33-86) was cloned into the pGEX4T vector and expressed as a GST-tagged protein. cDNA encoding mouse VAMP1, VAMP3, rat VAMP7 and VAMP8 was kindly provided by C. Hu (Louisville, KY). They were cloned into modified pcDNA3.1 vectors with the HA tag fused to their C-terminus. The construct encoding the His6-tagged sortase (SrtA*) was kindly provided by B. Pentelute (Boston, MA) and has been described previously ⁵¹ .

Bioinformatics: Searched the Uniprot database with JackHMMER on the HMMER web server using the BoNT type A sequence (Uniprot accession number A5HZZ9) until convergence. The returned sequences were aligned with Clustal Omega and the NeighborNet phylogenetic network evaluated with SplitsTree4.

Protein purification: E. coli BL21 (DE3) was used for protein expression. Expression induction was performed with 0.1 mM IPTG at 22° C. overnight. Bacterial pellets were disrupted in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl) by sonication and supernatants were collected after centrifugation at 20,000 g for 30 min at 4°C. Protein purification was performed using the AKTA Prime FPLC system (GE) and the purified proteins were further desalted with a PD-10 column (GE, 17-0851-01). In particular, inactive full length BoNT/X (BoNT/XRY) was cloned into the pET22b vector. The appropriate plasmid was transformed into E. coli BL21 (DE3) competent cells. The resulting colonies were used to inoculate 100 ml overnight cultures in TB medium containing 100 μg/ml carbenicillin in a 250 ml shake flask and grown at 37°C. Expression cultures were first grown using a LEX Bioreactor (Epiphyte3, Ontario, Canada) at 37° C. in 1.5 L of TB medium until an OD ₆₀₀ of 0.8 was reached. The temperature was then lowered to 18°C to induce expression with 1 mM IPTG and grown for 16-17 h. Cells were harvested and resuspended on ice in 50 mM HEPES, pH 7.2, 500 mM NaCl, 25 mM imidazole, 5% glycerol, 2 mM TCEP to ensure cell lysis with Emulsiflex-C3 (Avestin, Mannheim, German) at 20,000 psi. inch. The lysate was ultracentrifuged at 200,000 g for 45 min at 4°C. The supernatant was loaded onto a 15 ml Protino® Ni-NTA agarose column (Macherey-Nagen, Duren, Germany), which was then washed with 50 mM HEPES, pH 7.2, 500 mM NaCl, 100 mM imidazole, 5% glycerol, 1 mM TSER. Elution was performed with 50 mM HEPES, pH 7.2, 500 mM NaCl, 250 mM imidazole, 5% glycerol, 1 mM TCEP. The eluate was dialyzed overnight in 50 mM HEPES, pH 7.2, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP at 4°C. The dialysate was concentrated using a Vivaspin concentrator (100 kDa cutoff, GE Healthcare, Danderyd, Sweden) before loading onto a Superdex200-16/60 column (GE Healthcare, Danderyd, Sweden) pre-equilibrated with the same buffer used for dialysis. The elution peak corresponding to BoNT/X was collected and concentrated so that the final sample had a concentration of 10 mg/ml. The sample was aliquoted and flash frozen in liquid nitrogen for storage at -80°C.

Ganglioside Binding Assay: Purified gangliosides GD1a, GD1b, GT1b and GM1 (Carbosynth, Compton, UK) were dissolved in DMSO and diluted with methanol to a final concentration of 2.5 μg/mL; 100 μl was added to each well of a 96-well PVC assay plate (catalog # 2595, Corning; Corning, NY). After evaporation of the solvent at 21° C., the wells were washed with 200 μl PBS/0.1% (w/v) BSA. Non-specific binding sites were blocked by incubation for 2.5 hours at 4° C. in 200 μl PBS/2% (w/v) BSA. Binding assays were performed in 100 µl PBS/0.1% (w/v) BSA per well for 1 h at 4°C with samples (in triplicate) at concentrations ranging from 6 to 0.05 µM ( in serial 2-fold dilution). After incubation, wells were washed three times with PBS/0.1% (w/v) BSA and incubated with HRP-conjugated anti-6xHis monoclonal antibody (1:2000, ThermoFisher) for 1 h at 4°C. After three washes with PBS/0.1% (w/v) BSA, bound samples were detected using Ultra-TMB (100 μl/well, ThermoFisher) as substrate. The reaction was stopped after 15 min by adding 100 μl of 0.2 M H ₂ SO ₄ and absorbance was measured at 450 nm using an Infinite M200PRO plate reader (Tecan, Mannedorf, Switzerland). Data was analyzed with Prism7 (GraphPad software).

Cleavage of SNARE Proteins in Rat Brain Surfactant (BDE) Extracts: Rat BDEs were prepared from freshly dissected adult rat brains as described previously ⁵⁸ . Briefly, the rat brain was homogenized in 15 ml of 320 mm sucrose buffer, and then centrifuged at 5000 rpm for 2 min at 4°C. The supernatants were collected and centrifuged at 11,000 rpm for 12 minutes. The precipitate was collected and solubilized for 30 min in 15 ml Tris buffered saline (TBS: 20 mM Tris, 150 mM NaCl) plus 2% Triton X-100 and coc

- 77 041568 protease inhibitors (Roche, CA). The samples were then centrifuged at 17,000 rpm for 20 minutes to remove insoluble materials. The final concentration of BDE is ~2 mg/ml of proteins. BDE (60 μl) were incubated with X-LC (0.5 μM), A-LC (1 μM) or B-LC (1 μM), respectively, for 1 h at 37°C and then analyzed by immunoblotting using enhanced chemiluminescence (ECL) method (Pierce). As controls, LCs were pre-incubated with 20 mM EDTA for 20 min at room temperature (RT) to deactivate their activity before adding to BDE.

Cleavage of recombinant VAMP by X-LC: VAMP2 (1-96) was expressed and purified as a His6-tagged protein and VAMP2 (33-86) was expressed and purified as a GST-tagged protein. These proteins (0.6 mg/ml) were incubated with 0.1 μM X-LC in TBS buffer for 1 hour at 37°C. Samples were either analyzed by SDS-PAGE gel and Coomassie blue staining or subjected to mass spectrometry analysis.

Cleavage of VAMP in cell lysates: Full length HA-labeled VAMP1, 3, 7 and 8 were transfected into HEK293 cells using PolyJet transfection reagents (SignaGen, MD) according to the manufacturer's instructions. Cell lysates were harvested 48 h later in RLPA buffer (50 mM Tris, 1% NP40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 400 µl per 10 cm dish) plus a protease inhibitor cocktail (Sigma- Aldrich). Cell lysates (250 µl) were incubated with X-LC (0.5 µM) for 1 h at 37°C. The samples were then analyzed by immunoblotting.

Whole Protein Analysis by LC-MS/MS: Samples were analyzed at the Taplin Specialty Center for Biological Mass Spectrometry at Harvard Medical School. Briefly, whole protein samples were loaded onto a 100 µm id C18 reverse phase HPLC column packed with 3 cm beads offline using a pressure cell. The column was reattached to an Accela 600 pump (Thermo Fisher Scientific, Waltham, MA). A fast ascending acetonitrile gradient was used to elute the protein/peptide from the HPLC column. As the peptides eluted, they were electrospray ionized and then injected into an LTQ Orbitrap Velos Pro ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) to perform a high resolution FTMS scan at 60,000 resolution, a second low resolution scan at ion trap and final scan to perform data dependent MS/MS. Envelopes in the charged state were removed by hand to obtain monoisotopic masses, when possible, or average masses for proteins. The identities of peptides and proteins were determined by matching protein databases to the resulting fragmentation pattern using Sequest software (Thermo Fisher Scientific). All databases contain a reverse version of all sequences, and the data was filtered to a false positive peptide detection rate of 1 to 2%.

Identification of a protease cleavage site between LC and Hn: A His6-tagged recombinant X-LC-HN fragment (residues 1-891) was purified in E. coli and subjected to limited proteolysis with Lys-C endoproteinase (Sigma P2289, molar ratio 100:1 ( toxin: Lys-C), 25 min at room temperature). The cleavage site was determined by Tandem Mass Tag (TMT) and a tandem mass spectrometry approach. Briefly, intact X-LC-HN samples were labeled with mild TMT and an equal number of X-LC-H _N samples were subjected to Lys-C followed by heavy TMT labeling. Both samples were then digested with chymotrypsin, pooled together, and subjected to quantitative analysis by mass spectrometry.

Cysteine alkylation with NEM: Lys-C activated X-LC-HN fragment was diluted in sodium phosphate buffer (10 mM, pH 6.5) at a final concentration of 0.3 mg/mL, with or without NEM at the indicated concentrations (20, 10 and 5 mM) and incubated for 10 min at RT. NEM was received fresh in sodium phosphate buffer. Samples were mixed with 3x neutral Loading Dye (200 mM Tris, pH 6.8, 30% glycerol, 6% lithium dodecyl sulfate, 10 mM NEM, and 0.06% BBB). For samples without NEM, the same 3x SDS Loading Dye without NEM was used. Samples were further incubated with Loading Dye at RT for 10 min, heated for 10 min at 55°C and then analyzed by SDS-PAGE and Coomassie blue staining.

Neuronal culture and immunoblotting analysis: Primary rat cortical neurons were prepared from E18-19 embryos using a papain dissociation kit (Worthington Biochemical, NJ) as previously described by the present inventors ⁵⁸ . Experiments were performed in DIV 14-16. Neurons were exposed to BoNT/X fragments or sortase-mediated ligation mixture in medium for 12 h. Cells were then washed and lysed with RIPA buffer (50 mM Tris, 1% NP40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS) plus a protease inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged for 10 min at maximum speed using a microcentrifuge at 4°C. Supernatants were subjected to SDS-PAG and immunoblot analysis.

Dot blot: BoNT (0.2 μg in 1 μl) was spotted onto nitrocellulose membranes and dried (10 min at room temperature). The membranes were blocked with 5% milk in TBST (TBS plus 0.05% Tween20) for 30 minutes and then incubated with the indicated antiserum (1:500 dilution) for 30 minutes. The membranes were then washed three times with TBST and incubated with HRP-conjugated

- 78 041568 (horseradish peroxidase) secondary antibodies for 30 min, washed three more times with

TBST and analyzed by the ECL method (Pierce). It should be noted that the BoNT/X sample consisted of purified X-LC-H _n and X-H _c in a ratio of 1:1.

Sortase-mediated ligation: GST-X-HC was digested overnight at 4°C with thrombin before being added to the protein mixture. The ligation reaction was carried out in 50 µl of TBS buffer with the addition of X-LC-HN (8 µM), thrombin-cleaved GST-X-HC (25 µM), Ca ²⁺ (10 mM), and sortase (10 µM) for 40 min at R.T. In FIG. 4C neurons were exposed to 5 μl of the mixture in the medium for 12 hours. In the DAS assay described in FIG. 4D, 25 μl of the mixture was administered to mice by injection into the hind limb.

DAS Assay: The sortase-mediated ligation mixture was first activated by limited proteolysis using trypsin (60:1 molar ratio (total proteins: trypsin), 30 min at RT). Trypsin was chosen over Lys-C because they could stop proteolysis by adding a trypsin inhibitor (soybean trypsin inhibitor, ratio 1:10 (trypsin: trypsin inhibitor). Mice (CD-1 strain, 21-25 g, n=6) were anesthetized with isoflurane ( 3-4%) and injected the sortase-mediated ligation mixture using a 30 gauge needle attached to sterile Hamilton syringes into the calf muscles of the right hind limb. h after injection as previously described ^{52, 53} .

- 79 041568

Links

1. Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis. Physiol Rev 80, 717-766 (2000).

2. Montal, M. Botulinum neurotoxin: a marvel of protein design. Annu Rev Biochem Ί9, 591-617 (2010).

3. Rossetto, 0., Pirazzini, M. & Montecucco, C. Botulinum neurotoxins: genetic, structural and mechanistic insights. Nat Rev Microbiol 12, 535-549 (2014).

4. Arnon, S.S., et al. Botulinum toxin as a biological weapon: medical and public health management. Jama 285, 1059-1070 (2001).

5. Pirazini, M., et al. Thioredoxin and its reductase are present on synaptic vesicles, and their inhibition prevents the paralysis induced by botulinum neurotoxins. Cell Rep 8, 18701878 (2014).

6. Pirazzini, M., et al. The thioredoxin reductase—Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles. Toxicon 107, 32-36 (2015).

7. JaHN, R. & Scheller, R.H. SNAREs—engines for membrane fusion. Nat Rev Mol Cell Biol 7, 631-643 (2006).

8. Sudhof, T.C. & Rothman, J.E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474-477 (2009).

9 Gu, S., et al. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science 335, 977-981 (2012).

10. Lee, K., et al. Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex. Science 344, 1405-1410 (2014).

11. Lee, K., et al. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoSPathog 9, el003690 (2013).

12. Sugawara, Y., et al. Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin. J Cell Biol 189, 691-700 (2010).

13. Hill, K.K., Xie, G., Foley, B.T. & Smith, T.J. Genetic diversity within the botulinum neurotoxin-producing bacteria and their neurotoxins. Toxicon 107, 2-8 (2015).

14. Johnson, E.A. Clostridial toxins as therapeutic agents: benefits of nature's most toxic proteins. Annu Rev Microbiol 53, 551-575 (1999).

15. Aoki, K.R. Botulinum toxin: a successful therapeutic protein. Curr Med Chem 11, 3085-3092 (2004).

16. Montecucco, C. & Molgo, J. Botulinal neurotoxins: revival of an old killer. Curr Opin Pharmacol 5, 274-279 (2005).

17. Dolly, J.0., Lawrence, G.W., Meng, J. & Wang, J. Neuro-exocytosis: botulinum toxins as inhibitory probes and versatile therapeutics. Curr Opin Pharmacol 9, 326-335 (2009).

- 80 041568

18. Burke, G.S. Notes on Bacillus botulinum. JBacteriol 4, 555-570 551 (1919).

19. Gimenez, D.F. & Ciccarelli, A.S. Another type of Clostridium botulinum. Zentralbl Bakteriol Orig 215, 221-224 (1970).

20. Lange, 0., et al. Neutralizing antibodies and secondary therapy failure after treatment with botulinum toxin type A: much ado about nothing? Clin Neuropharmacol 32, 213218 (2009).

21. Dong, M., et al. SV2 is the protein receptor for botulinum neurotoxin A. Science 312, 592-596 (2006).

22. Dong, M., et al. Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells. J Cell Biol 162, 1293-1303 (2003).

23. Mahrhold, S., Rummel, A., Bigalke, H., Davletov, B. & Binz, T. The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves. FEBS Lett 580, 2011-2014(2006).

24. Nishiki, T., et al. Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes. J Biol Chem 269, 10498-10503 (1994).

25 Schiavo, G., et al. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, 832-835 (1992).

26 Schiavo, G., et al. Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBSLett 335, 99-103 (1993).

27. Blasi, J., et al. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365, 160-163 (1993).

28. Smith, T.J., et al. Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization. Infect Immun 73, 5450-5457 (2005).

29. Hill, K.K., et al. Genetic diversity among Botulinum Neurotoxin-producing clostridial strains. JBacteriol 189, 818-832 (2007).

30. Montecucco, C. & Rasotto, M.B. On botulinum neurotoxin variability. MBio 6(2015).

31. Dover, N., Barash, J.R., Hill, K.K., Xie, G. & Amon, S.S. Molecular characterization of a novel botulinum neurotoxin type H gene. J Infect Dis 209, 192-202 (2014).

32 Barash, J.R. & Arnon, S.S. A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins. J Infect Dis 209, 183-191 (2014).

33. Maslanka, S.E., et al. A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin. J Infect Dis (2015).

34. Kalb, S.R., et al. Functional characterization of botulinum neurotoxin serotype H as a hybrid of known serotypes F and A (BoNT F/A). Anal Chem 87, 3911-3917 (2015).

- 81 041568

35. Luquez, C., Raphael, B. H. & Maslanka, S.E. Neurotoxin gene clusters in Clostridium botulinum type Ab strains. Appl Environ Microbiol 75, 6094-6101 (2009).

36. Dover, N., et al. Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: bont/A2, bont/F4 and bont/F5. PLoS One 8, e61205 (2013).

37. Dabritz, H.A., et al. Molecular epidemiology of infant botulism in California and elsewhere, 1976-2010. J Infect Dis 210, 1711-1722(2014).

38. Franciosa, G., Ferreira, J.L. & Hatheway, C.L. Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. J Clin Microbiol 32, 1911-1917 (1994).

39. Hutson, R.A., et al. Genetic characterization of Clostridium botulinum type A containing silent type B neurotoxin gene sequences. J Biol Chem 271, 10786-10792 (1996).

40. Kakinuma, H., Maruyama, H., Takahashi, H., Yamakawa, K. & Nakamura, S. The first case of type B infant botulism in Japan. Acta Paediatr Jpn 38, 541-543 (1996).

41. Kozaki, S., et al. Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan. Infect Immun 66, 4811-4816 (1998).

42. Ihara, H., et al. Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity. Biochim Biophys Acta 1625, 19-26 (2003).

43. Rummel, A., Bade, S., Alves, J., Bigalke, H. & Binz, T. Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity. J Mol Biol 326, 835-847 (2003).

44. Rummel, A., Mahrhold, S., Bigalke, H. & Binz, T. The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction. Mol Microbiol 51, 631-643 (2004).

45. Rossetto, 0., et al. SNARE motif and neurotoxins. Nature 372, 415-416 (1994).

46. Masuyer, G., Beard, M., Cadd, V.A., Chaddock, J.A. & Acharya, K.R. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B. J Struct Biol 174, 52-57 (2011).

47 Chaddock, J.A., et al. Expression and purification of catalytically active, nontoxic endopeptidase derivatives of Clostridium botulinum toxin type A. Protein Expr Purif 25, 219-228 (2002).

48. Ewbank, J.J. & Creighton, T.E. The molten globule protein conformation probed by disulphide bonds. Nature 350, 518-520 (1991).

- 82 041568

49. Nagy, P. Kinetics and mechanisms of thiol-disulfide exchange covering direct substitution and thiol oxidation-mediated pathways. Antioxid Redox Signal 18, 1623-1641 (2013).

50. Popp, M.W., Antos, J.M., Grotenbreg, G.M., Spooner, E. & Ploegh, H.L. Sorting: a versatile method for protein labeling. Nat Chem Biol 3, 707-708 (2007).

51. McCluskey, A.J. & Collier, R.J. Receptor-directed chimeric toxins created by sortase-mediated protein fusion. Mol Cancer Ther 12, 2273-2281 (2013).

52 Broide, R.S., et al. The rat Digit Abduction Score (DAS) assay: a physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis. Toxicon 71, 18-24 (2013).

53. Aoki, K.R. A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice. Toxicon 39, 1815-1820 (2001).

54. Binz, T., Bade, S., Rummel, A., Kollewe, A. & Alves, J. Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization. Biochemistry Al, 1717-1723 (2002).

55 Fu, Z., et al. Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate. Biochemistry 45, 8903-8911 (2006).

56. Pier, C.L., et al. Recombinant holotoxoid vaccine against botulism. Infect Immun 76, 437-442 (2008).

57. Mootz, H.D. Split inteins as versatile tools for protein semisynthesis. Chembiochem 10, 2579-2589 (2009).

58. Peng, L., Tepp, W.H., JoHNSon, E.A. & Dong, M. Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors. PLoSPathog 7, el002008 (2011).

59. Steegmaier, M., Klumperman, J., Foletti, D.L., Yoo, J.S. & Scheller, R.H. Vesicle-associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking. Mol Biol Cell IQ, 1957-1972 (1999).

60. Brandhorst, D., et al. Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity. Proc Natl Acad Sci USA 103, 2701-2706 (2006).

61. Raingo, J., et al. VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission. Nat Neurosci 15, 738-745 (2012).

62. Cocucci, E., Racchetti, G., Rupnik, M. & Meldolesi, J. The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4. J Cell Sci 121, 2983-2991 (2008).

63. Nicholson-Fish, J.C., Kokotos, A.C., Gillingwater, T.H., Smillie, K.J. & Cousin, M. A. VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis. Neuron 88, 973-984 (2015).

- 83 041568

64 Zeng, Q., et al. A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane. Mol Biol Cell 9, 2423-2437 (1998).

65. Krzewski, K., Gil-Krzewska, A., Watts, J., Stern, J.N. & Strominger, J.L.

VAMP4- and VAMP7-expressing vesicles are both required for cytotoxic granule exocytosis in

NK cells. Eur J Immunol 41, 3323-3329 (2011).

66. Yamamoto, H., et al. Specificity of botulinum protease for human VAMP family proteins. Microbiol Immunol 56, 245-253 (2012).

67. McNew, J.A., et al. Ykt6p, a prenylated SNARE essential for endoplasmic reticulum-Golgi transport. J Biol Chem 272, 17776-17783 (1997).

68. Kweon, Y., Rothe, A., Conibear, E. & Stevens, T.H. Ykt6p is a multifunctional yeast R-SNARE that is required for multiple membrane transport pathways to the vacuole. Mol Biol Cell 14, 1868-1881 (2003).

69. Hasegawa, H., et al. Mammalian ykt6 is a neuronal SNARE targeted to a specialized compartment by its profilin-like amino terminal domain. Mol Biol Cell 14, 698-720 (2003).

70. Daste, F., Galli, T. & Tareste, D. Structure and function of longin SNAREs. J

Cell Sci 128, 4263-4272 (2015).

71. Cooper, A.A., et al. Alpha-synuclein blocks ER-Golgi traffic and Rabi rescues neuron loss in Parkinson's models. Science 313, 324-328 (2006).

72. Thayanidhi, N., et al. Alpha-synuclein delays endoplasmic reticulum (ER)-to-

Golgi transport in mammalian cells by antagonizing ER/Golgi SNAREs. Mol Biol Cell 21, 18501863 (2010).

Other embodiments

All features disclosed in the present description may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a general series of equivalent or similar features.

From the above description, a person skilled in the art will be able to easily ascertain the main characteristics of the present disclosure and, without departing from its spirit and scope, will be able to make various changes and modifications to the present disclosure in order to adapt it to various applications and conditions. Thus, other embodiments also fall within the scope of the claims.

Equivalents and volume

While some embodiments of the present invention have been described and illustrated herein, a number of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the benefits described herein will be apparent to those skilled in the art. and each of such variations and/or modifications fall within the scope of the embodiments of the present invention described herein. In general, those skilled in the art will readily recognize that all parameters, measurements, materials, and configurations described herein are typical, and that the actual parameters, measurements, materials, and/or configurations will depend on the particular application or applications for which use the ideas of the present invention. Those skilled in the art will be able to recognize or ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced otherwise than as specifically described and claimed. Embodiments of the present invention are directed to each individual feature, system, product, material, kit and/or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits and/or methods, unless such features, systems, articles, materials, kits and/or methods are mutually incompatible, is included within the scope of the invention in accordance with real disclosure.

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Claims (20)

It is to be understood that all definitions defined and used herein control dictionary definitions, definitions in documents incorporated by reference, and/or conventional meanings of the defined terms. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the claimed subject matter for which each is cited, and in some cases the reference may cover the entire document. The use herein of objects in the singular in the description and claims, unless expressly stated otherwise, should be understood as at least one. The phrase and/or as used herein in the description and claims is to be understood as one or both of the elements so connected, i.e. elements that in one case are present together, and in other cases separately. Several elements listed with and/or should be interpreted in the same way, i.e. one or more elements connected in this way. Other elements may optionally be present, other than those specifically specified by the condition and/or whether or not they are related to those elements specifically defined. Thus, as a non-limiting example, a reference to A and/or B, when used together in a public phrase such as containing, may, in one embodiment, refer to only A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in one embodiment, both A and B (optionally including other elements); and so on. Used herein in the description and in the claims, the union or should be understood as having the same meaning as the union and/or defined above. For example, when separating elements in a list, the union or or and/or should be interpreted as inclusive, i.e. including at least one, but also including more than one of a number or list of elements, and optionally additional elements not listed. Only terms clearly marked as opposites, such as only one of, or exclusively one of, or when used in a claim, consisting of, will refer to the inclusion of exactly one element from a series or list of elements. As a general rule, the term or as used herein should only be construed as indicating exclusive alternatives (i.e. one or the other, but not both) when preceded by terms of exclusivity such as either, one of, only one of, or exclusively one of. The term consisting essentially of used in the claims has its usual meaning used in the field of patent law. As used herein in the description and claims, the phrase at least one in relation to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but optionally includes at least one of each element specifically listed in the list of elements and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically defined in the list of elements to which the phrase at least one refers may optionally be present, whether or not they are related to those elements specifically defined. Thus, as a non-limiting example, at least one of A and B (or, equivalently, at least one of A or B, or equivalently, at least one of A and/or B) can be related according to one variant implementing at least one, optionally including more than one, A, without the presence of B (and optionally including elements other than B); according to another embodiment, at least one, optionally including more than one B, without the presence of A (and optionally including elements other than A); in another embodiment, at least one, optionally including more than one A and at least one, optionally including more than one B (and optionally including other elements); and so on. It should also be appreciated that, unless expressly stated to the contrary, in any methods claimed herein that involve more than one step or action, the order of the steps or steps of the method is not necessarily limited to the order in which the steps or steps of the method are set forth. In the claims, as well as in the above description, all transitional phrases such as containing, including, carrying, having, including, consisting of and the like, should be understood as open, i.e. meaning including without limitation. Only passphrases consisting of and consisting essentially of are closed or semi-closed passphrases, respectively, as specified in the Patent Examining Procedures Manual of the US Patent Office, Section 2111.03. CLAIM 1. An isolated Clostridial botulinum neurotoxin (BoNT) polypeptide containing an amino acid sequence characterized by at least 85%, at least 86%, - 85 041568 at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with respect to any of SEQ ID NO: 1-3. 2. An isolated BoNT polypeptide according to claim 1, containing the amino acid sequence of any of SEQ ID NOs: 1-3. 3. An isolated BoNT polypeptide according to claim 1, containing one or more substitution type mutations at the position corresponding to C461, C467 or C1240 in SEQ ID NO: 1. 4. An isolated BoNT polypeptide according to claim 1, containing a single substitution type mutation at the position corresponding to C461 or C467 of SEQ ID NO: 2. 5. An isolated BoNT polypeptide according to claim 1, comprising the amino acid sequence of any one of SEQ ID NOs: 22-24, wherein said polypeptide is a chimeric BoNT peptide. 6. An isolated BoNT polypeptide according to claim 1, containing: (a) a protease domain; (b) a modified linker site; (c) a translocation domain, wherein (a), (b) and (c) are from BoNT serotype X, and wherein the modified linker region contains one single substitution type mutation at the position corresponding to C461 or C467 of SEQ ID NO: 1. 7. An isolated BoNT polypeptide according to claim 6, further comprising (d) a receptor binding domain. 8. An isolated BoNT polypeptide according to claim 7, wherein the receptor binding domain is from BoNT/X. 9. An isolated BoNT polypeptide according to claim 8, wherein the receptor binding domain contains a substitution mutation corresponding to C1240S or C1240A in SEQ ID NO: 1. 10. An isolated BoNT polypeptide according to claim 7, wherein the receptor binding domain is from a serotype selected from the group consisting of A, B, C, D, E, F, and G. 11. An isolated BoNT polypeptide according to claim 6, wherein the modified linker region contains an artificial linker. 12. An isolated BoNT polypeptide according to claim 11, wherein the artificial linker contains a protease cleavage site. 13. An isolated BoNT polypeptide according to claim 1, containing one or more substitution type mutations at the position corresponding to R360, Y363, H227, E228 or H231 in SEQ ID NO: 1. 14. An isolated BoNT polypeptide according to claim 13 containing a mutation at the position corresponding to R360 and Y363 of SEQ ID NO: 1. 15. The isolated BoNT polypeptide according to claim 13, containing the amino acid sequence of SEQ ID NOs: 31-38. 16. An isolated BoNT polypeptide according to claim 1, wherein the amino acid sequence is not 100% identical to SEQ ID NOs: 1-3. 17. A chimeric molecule comprising a first moiety linked to a second moiety, wherein the first moiety is the isolated BoNT polypeptide of claim 13. 18. A method of treating a condition, the method comprising administering a therapeutically effective amount of an isolated BoNT polypeptide according to any one of claims 1-16, or a chimeric molecule according to claim 17, to a subject for treating a condition, the condition being associated with overactive neurons or glands. 19. A method for producing a BoNT polypeptide, wherein the method comprises: (i) obtaining a first BoNT fragment consisting of a light chain (LC) and an N-terminal heavy chain domain (H _N ), wherein the first BoNT fragment contains the C-terminal LPXTGG motif (SEQ ID NO: 60); (ii) obtaining a second BoNT fragment consisting of a heavy chain C-terminal domain (H _C ), wherein the second BoNT fragment contains a specific protease cleavage site at its N-terminus; (iii) cleavage of the second BoNT fragment with a specific protease, wherein the cleavage results in a free glycine residue at the N-terminus; and (iv) contacting the first BoNT fragment and the second BoNT fragment in the presence of a transpeptidase, thereby ligating the first BoNT fragment and the second BoNT fragment to form a ligated BoNT. 20. Selected polypeptide neurotoxin Clostridial botulinum serotype X (BoNT/X), containing a light chain and a heavy chain, where L _C contains the amino acid sequence of SEQ ID NO: 3; where the heavy chain contains an amino acid sequence expressed as amino acids -