NOVEL BOTULINUM NEUROTOXIN AND ITS DERIVATIVES
Provided herein are Clostridial Botulinum neurotoxin (BoNT) polypeptides of a novel serotype (BoNT/X) and methods of making and using the BoNT polypeptides, e.g., in therapeutic applications.
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This application claims the benefit under 35 U.S.C. § 119(c) of U.S. provisional application No. 62/360,239, filed Jul. 8, 2016, which is incorporated by reference herein in its entirety.
GOVERNMENT SUPPORTThis invention was made with government support under R01NS080833 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUNDClostridial Botulinum neurotoxins (BoNTs) are among the most dangerous potential bioterrorism agents and are also used clinically to treat a growing list of medical conditions. There are seven serotypes of BoNTs (BoNT/A-G) known to date. In recent years, BoNTs have been widely used to treat a growing list of medical conditions: local injections of minute amount of toxins can attenuate neuronal activity in targeted regions, which can be beneficial in many medical conditions as well as for cosmetic purposes. As the application of BoNTs grows, limitations and adverse effects have been reported. The major limitation is the generation of neutralizing antibodies in patients, which renders future treatment ineffective. Termination of BoNT usage often leaves patients with no other effective ways to treat/relieve their disorders. Adverse effects associated with BoNT use range from transient non-serious events such as ptosis and diplopia to life-threatening events even death. The limitations and adverse effects of BoNTs are largely correlated with dose. There are considerable interests in developing novel BoNT types as therapeutic toxins. No new BoNT types have been recognized for the past 45 years.
SUMMARYThe present disclosure is based, at least in part, on the identification of a novel BoNT serotype, BoNT/X, from searching genomic database of Clostridium Botulinum strains. BoNT/X the lowest sequence identity with other BoNTs and it is not recognized by antisera raised against known BoNT types. BoNT/X cleaves SNARE proteins, like other BoNTs. However, BoNT/X also cleave several SNARE proteins that other BoNTs cannot cleave, e.g., VAMP4, VAMP5, and Ykt6. Compositions and methods for treating diseases using BoNT/X are provided. Also provided herein are methods of making BoNT/X.
Accordingly, some aspects of the present disclosure provide isolated Clostridial Botulinum neurotoxin (BoNT) polypeptides comprising the amino acid sequence of SEQ ID NO: 1.
Some aspects of the present disclosure provide isolated BoNT polypeptides comprising an amino acid sequence that has 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 to SEQ ID NO: 1. In some embodiments, the isolated BoNT polypeptide consists of the amino acid sequence of SEQ ID NO: 1.
Some aspects of the present disclosure provide isolated BoNT polypeptides comprising the amino acid sequence of SEQ ID NO: 2. Some aspects of the present disclosure provide isolated BoNT polypeptides an amino acid sequence that has 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 to SEQ ID NO: 2. In some embodiments, the isolated BoNT polypeptide consists of the amino acid sequence of SEQ ID NO: 2.
Some aspects of the present disclosure provide isolated BoNT polypeptides comprising the amino acid sequence of SEQ ID NO: 3. Some aspects of the present disclosure provide isolated BoNT polypeptides an amino acid sequence that has 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 to SEQ ID NO: 3. In some embodiments, the isolated BoNT polypeptide consists of the amino acid sequence of SEQ ID NO: 3.
Some aspects of the present disclosure provide modified BoNT polypeptides comprising one or more substitution mutation(s) in a position corresponding to C461, C467, and C1240 of 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, or C467A/C1240A in SEQ ID NO: 1.
In some embodiments, the modified BoNT polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 4-17. In some embodiments, the modified BoNT polypeptide comprises an amino acid sequence that has 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 to any of SEQ ID NOs: 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 one of SEQ ID NOs: 4-17.
Some aspects of the present disclosure provide modified BoNT polypeptides comprising a single substitution mutation in a position corresponding to C461 or C467 of 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, C461S/C1240A, C467A/C1240S, or C467A/C1240A in SEQ ID NO: 2.
In 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 comprises an amino acid sequence that has 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 to any of SEQ ID NOs: 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 one of SEQ ID NOs: 18-21.
Some aspects of the present disclosure provide chimeric BoNT polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 22-24. In some embodiments, the chimeric BoNT polypeptide comprises an amino acid sequence that has 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 to any one of SEQ ID NOs: 22-24, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In 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 in a position corresponding to C461 or C467 of in SEQ ID NO: 2.
In 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 has 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 to any one of SEQ ID NOs: 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 some embodiments, the BoNT polypeptide enters a cell. In some embodiments, the BoNT polypeptide cleaves a 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. In some embodiments, the BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 39.
In some embodiments, the SNARE protein is VAMP2. In some embodiments, the BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 40.
In some embodiments, the SNARE protein is VAMP3. In some embodiments, the 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, the BoNT cleaves between amino acid residues corresponding to K87 and S88 of SEQ ID NO: 42.
In some embodiments, the SNARE protein is VAMP5. In some embodiments, the BoNT cleaves between amino acid residues corresponding to R40 and S41 of SEQ ID NO: 43.
In some embodiments, the SNARE protein is Ykt6. In some embodiments, the 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 compared to its corresponding wild type BoNT polypeptide.
In some embodiments, the cell is a secretory cell. In some embodiments, the cell is a neuronal cell. In some embodiments, the cell is an immune cell. In some embodiments, the BoNT polypeptide suppresses neuronal activity. In some embodiments, the BoNT polypeptide induces flaccid paralysis. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is from a mammal. In some embodiments, the mammal is a human. In some embodiments, mammal is a rodent. In some embodiments, the rodent is a mice. In 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.
Other aspects of the present disclosure provide nucleic acid molecules comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 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 BoNT polypeptide described herein. Nucleic acid vectors comprising such nucleic acid molecules are provided. Cells comprising the nucleic acid molecules or the nucleic acid vectors described herein are provided. In some embodiments, such cells express the BoNT polypeptide described herein.
Methods of producing the BoNT polypeptide of the present disclosure are provided. Such methods comprise the steps of culturing the cell expressing the BoNT polypeptides under conditions wherein said BoNT polypeptide is produced. In some embodiments, the methods further comprise recovering the BoNT polypeptide from the culture.
Other aspects of the present disclosure provide modified BoNT polypeptides comprising: (a) a protease domain; (b) a modified linker region; and (c) a translocation domain; wherein (a), (b), and (c) are from BoNT serotype X, and wherein the modified linker region comprises one single substitution mutation in a 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, modified linker region comprises a substitution mutation corresponding to C461S or C461A in SEQ ID NO: 1. In some embodiments, the modified linker region comprises a substitution mutation corresponding to C467S or C467A in SEQ ID NO: 1.
In some embodiments, the receptor binding domain is from BoNT/X. In some embodiments, the receptor binding domain is modified. In some embodiments, the receptor binding domain comprises a substitution mutation corresponding to C1240S or C1240A in SEQ ID NO: 1.
In some embodiments, the receptor binding domain is from a serotype selected from the group consisting of A, B, C, D, E, F, and G.
In some embodiments, the modified BoNT polypeptide enters a 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. In some embodiments, the BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 39.
In some embodiments, the SNARE protein is VAMP2. In some embodiments, the BoNT cleaves between amino acid residues corresponding to R66 and A67 of SEQ ID NO: 40.
In some embodiments, the SNARE protein is VAMP3. In some embodiments, the 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, the BoNT cleaves between amino acid residues corresponding to K87 and S88 of SEQ ID NO: 42.
In some embodiments, the SNARE protein is VAMP5. In some embodiments, the BoNT cleaves between amino acid residues corresponding to R40 and S41 of SEQ ID NO: 43.
In some embodiments, the SNARE protein is Ykt6. In some embodiments, the 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 compared to its corresponding wild type BoNT polypeptide.
In some embodiments, the cell is a secretory cell. In some embodiments, the cell is a neuronal cell. In some embodiments, the cell is an immune cell. In some embodiments, the BoNT polypeptide suppresses neuronal activity. In some embodiments, the BoNT polypeptide induces flaccid paralysis. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is from a mammal. In some embodiments, the mammal is a human. In some embodiments, mammal is a rodent. In some embodiments, the rodent is a mice. In 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 some embodiments, the modified linker region comprises an artificial linker. In some embodiments, the artificial linker contains a cleavage site of a protease. 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 some embodiments, the linker comprises the amino acid sequence of any of SEQ ID NOs: 50-60).
Other aspects of the present disclosure provide nucleic acid molecules comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 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 BoNT polypeptide described herein. Nucleic acid vectors comprising such nucleic acid molecules are provided. Cells comprising the nucleic acid molecules or the nucleic acid vectors described herein are provided. In some embodiments, such cells express the BoNT polypeptide described herein.
Methods of producing the BoNT polypeptide of the present disclosure are provided. Such methods comprise the steps of culturing the cell expressing the BoNT polypeptides under conditions wherein said BoNT polypeptide is produced. In some embodiments, the methods further comprise recovering the BoNT polypeptide from the culture.
Other aspects of the present disclosure provide modified BoNT polypeptides comprising one or more substitution mutation(s) in positions corresponding to R360, Y363, H227, E228, or H231 in SEQ ID NO: 1. In some embodiments, the one or more substitution mutation corresponds to R360A/Y363F, H227Y, E228Q, or H231Y in SEQ ID NO: 1.
In 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 comprises an amino acid sequence that has 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 to any of SEQ ID NOs: 31-38, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the modified BoNT polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 31-38.
Other aspects of the present disclosure provide modified BoNT/X polypeptide comprising: a) an inactive protease domain; b) a linker region; and c) a translocation domain. In some embodiments, the modified BoNT/X further comprises a receptor binding domain.
In some embodiments, the inactive protease domain comprises one or more substitution mutations in positions corresponding to R360, Y363, H227, E228, or H231 of SEQ ID NO: 1. In some embodiments, the 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 a cell. In some embodiments, the modified BoNT polypeptide does not cleave a SNARE protein.
In some embodiments, the modified BoNT/X polypeptide further comprises a modification in the linker region of (b). In some embodiments, the modification in the linker region comprises one single substitution mutation in a position corresponding to C461 or C467 of SEQ ID NO: 1. In some embodiments, the single substitution mutation corresponds to C461A, C461S, C467A, or C467S in SEQ ID NO: 1. In some embodiments, the modified BoNT/X polypeptide further comprises a modification in the receptor binding domain of (d).
In some embodiments, the modification in the receptor binding domain comprises a substitution mutation in a position corresponding to C1240 of SEQ ID NO: 1.
Other aspects of the present disclosure provide nucleic acid molecules comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 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 BoNT polypeptide described herein. Nucleic acid vectors comprising such nucleic acid molecules are provided. Cells comprising the nucleic acid molecules or the nucleic acid vectors described herein are provided. In some embodiments, such cells express the BoNT polypeptide described herein.
Methods of producing the BoNT polypeptide of the present disclosure are provided. Such methods comprise the steps of culturing the cell expressing the BoNT polypeptides under conditions wherein said BoNT polypeptide is produced. In some embodiments, the methods further comprise recovering the BoNT polypeptide from the culture.
Further provided herein are use of the modified BoNT polypeptide described herein as a delivery vehicle to deliver therapeutics into neurons.
Some aspects of the present disclosure provide chimeric molecules comprising a first portion linked to a second portion, wherein the first portion is a modified BoNT polypeptide described herein.
In some embodiments, the first portion and the second portion are linked covalently. In some embodiments, the first portion and the second portion are linked non-covalently.
In some embodiments, wherein the second portion is selected from the group consisting of a small molecule, a nucleic acid, a short polypeptide and a protein. In some embodiments, the second portion is a bioactive molecule. In some embodiments, the second portion is a non-polypeptide drug. In some embodiments, the second portion is a therapeutic polypeptide.
Other aspects of the present disclosure provide nucleic acid molecules comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 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 comprising such nucleic acid molecules are provided. Cells comprising the nucleic acid molecules or the nucleic acid vectors described herein are provided. In some embodiments, such cells express the chimeric BoNT polypeptide described herein.
Methods of producing the chimeric BoNT polypeptide of the present disclosure are provided. Such methods comprise the steps of culturing the cell expressing the chimeric BoNT polypeptides under conditions wherein said chimeric BoNT polypeptide is produced. In some embodiments, the methods further comprise recovering the chimeric BoNT polypeptide from the culture.
Other aspects of the present disclosure provide pharmaceutical compositions comprising the BoNT polypeptides described herein.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
Kit comprising such pharmaceutical compositions and directions for therapeutic administration of the pharmaceutical composition are also provided.
Some aspects of the present disclosure provide methods of treating a condition, comprising administering a therapeutically effective amount of the BoNT polypeptide, the chimeric molecule, or the pharmaceutical composition described herein to a subject to treat the condition.
In some embodiments, the condition is associated with overactive neurons or glands. In some embodiments, the condition is selected from the group consisting of, spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain from muscle spasms, headache pain, dermatological or aesthetic/cosmetic conditions, and obesity/reduced appetite.
In some embodiments, the condition is not associated with unwanted neuronal activity. In some embodiments, the condition is selected from the group consisting of: psoriasis, allergy, haemophagocytic lymphohistiocytosis, and alcoholic pancreatic diseases.
In some embodiments, the administering is via injection to where unwanted neuronal activity is present.
Yet other aspects of the present disclosure provide methods of producing a Clostridial Botulinum neurotoxin (BoNT) polypeptide, the method comprising:
(i) obtaining a first BoNT fragment comprising a light chain (LC) and a N-terminal domain of a heavy chain (HN), wherein the first BoNT fragment comprises a C-terminal LPXTGG (SEQ ID NO: 60) motif;
(ii) obtaining a second BoNT fragment comprising a C-terminal domain of the heavy chain (HC); wherein the second BoNT fragment comprise a specific protease cleavage site at its N-terminus;
(iii) cleaving 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 further comprises an affinity tag. In 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 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 some embodiments, the second BoNT fragment further comprises an affinity tag. In 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 the 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. In 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. In some embodiments, the second BoNT fragment is from BoNT/A. In some embodiments, the second BoNT fragment is from BoNT/B. In some embodiments, the second BoNT fragment is from BoNT/C. In some embodiments, the second BoNT fragment is from BoNT/X.
In some embodiments, the transpeptidase is a sortase. In some embodiments, the sortase is from Staphylococcus aureus (SrtA).
These and other aspects of the disclosure, as well as various advantages and utilities will be apparent with reference to the Detailed Description of the Invention. Each aspect of the disclosure can encompass various embodiments as will be understood.
All documents identified in this application are incorporated in their entirety herein by reference.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Clostridium Botulinum neurotoxins (BoNTs) are a family of bacterial toxins produced by clostridium bacteria, with seven well-established serotypes (BoNT/A-G) 1-3. They are one of the most dangerous potential bio-terrorism agents, classified as a “Category A” select agent by Center for Disease Control (CDC) of United States 4. 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 connected via an inter-chain disulfide bond. The He contains two sub-domains: the N-terminal HN domain that mediates translocation of the LC across endosomal membranes, and the C-terminal HC domain that mediates binding to receptors on neurons. The inter-chain disulfide bond is reduced once the LC translocates into the cytosol5,6. Released LC acts as a protease to specifically cleave a set of neuronal proteins: BoNT/A, C, and E cleave at distinct sites on a protein known as SNAP-25; BoNT/B, D, F, and G cleave at different sites on a vesicle protein VAMP; and BoNT/C also cleaves a transmembrane protein syntaxin 11-3. These three proteins form a complex, known as SNARE complex, which is essential for release of neurotransmitters7,8. Cleavage of any one of these three SNARE proteins blocks neurotransmitters release from neurons, thus paralyzing muscles.
BoNTs are the most potent toxins known and cause the human and animal disease known as botulism3. The major form of botulism is caused by ingesting food contaminated with BoNTs (food botulism). Other forms also exist such as infant botulism, which is due to colonization of the intestine by toxin-producing bacteria in infants. 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 BoNTs and protects BoNTs from proteases in the gastrointestinal tract9. Genes encoding BoNT and NTNHA are found in two types of gene clusters: (1) HA cluster, containing genes for three conserved proteins HA17, HA33 and HA70, which form a complex with BoNT/NTNHA and facilitate absorption of toxins across the intestinal epithelial barrier10-12 (2) OrfX cluster, which encodes conserved OrfX1, OrfX2, OrfX3 and P47 proteins with unknown functions13.
Because local injections of minute amounts of toxins can attenuate neuronal activity in targeted regions, BoNTs have been used to treat a growing list of medical conditions 141, including muscle spasms, chronic pain, overactive bladder problems, as well as for cosmetic applications. The market for BoNTs has already surpassed $1.5 billion in 2011 and is projected to reach 2.9 billion by 2018.
BoNTs were traditionally typed by neutralization assays in mice, by injecting culture supernatant of clostridium bacteria into mice, with or without antisera against known BoNTs. The first distinguished serotypes, BoNT/A and BoNT/B, were established in 1919 by Georgina Burke 18. The last of the seven type, BoNT/G, was recognized in 1969 from soil samples in Argentina 9. No new serotype of BoNTs has been recognized since 1970. This classification held true after protein sequences for each BoNT was determined in 1990's. The sequence identity between any two pairs among the seven BoNTs ranges from 32% to 65.3%. All seven BoNTs have been identified and characterized before the era of their medical use. Therefore, there is no patent on any of these toxins. Any company is free to produce and market any one of these seven BoNTs. Among the seven types, BoNT/A and BoNT/B are the two toxins that are currently FDA-approved for use in humans 1416 BoNT/A is the dominant type used for both medical and cosmetic applications, marketed as Botox from Allergan Inc., Dysport from IPSEN Inc., and Xeomin from Merz Inc. BoNT/B is marketed as Myobloc by USWorld Med. There are considerable interests in developing other BoNT types as therapeutic toxins, for two major reasons:
(1) A major limitation in treatment is generation of neutralizing antibody against BoNT/A or BoNT/B in patients, which renders future treatment with the same toxin ineffective20. In this case, patients will need to be treated with a different type of BoNTs. This is why BoNT/B is often utilized to treat patients who have generated neutralizing antibodies against BoNT/A during treatment, but there is a need for alternative toxins for patients who have generated antibodies against both BoNT/A and BoNT/B.
(2) Although all BoNTs share the same structure and function, there are also considerable differences between them. For instance, BoNT/A cleaves SNAP-25 and uses a protein SV2 as its receptor, whereas BoNT/B cleaves VAMP and uses a protein synaptotagmin (Syt) as its receptor21-27. These functional variations may translate to potential differences in therapeutic efficacy targeting distinct types of neurons. In addition, the stability and therapeutic duration can be also different among seven types of toxins. Therefore, a different toxin type may have its advantage over BoNT/A and BoNT/B.
Rapid progress on genomic sequencing in recent years has revealed a remarkable diversity of BoNTs28-29. First, there are multiple subtypes, which can be recognized by the same antiserum, but contain significant levels of variations on protein sequences (2.6%˜31.6% differences)28,30. For instance, BoNT/A contains 8 known subtypes, designated as BoNT/A1-A813. Furthermore, multiple mosaic toxins exist, likely derived from recombination of toxin genes. For instance, a “type H” was reported in 2013, but it was later recognized as a chimeric toxin because its LC shares˜80% identity with the LC of a BoNT/F subtype, BoNT/F5, and its HC shares ˜84% identity to the HC of BoNT/A131-34. Consistently, this toxin can be recognized and neutralized by available antisera against BoNT/A33.
The gene cluster encoding BoNTs can be on plasmids, bacterial phage, or chromosomes, indicating that the toxin genes are mobile and subject to horizontal gene transfer13. There are also cases that a clostridium bacteria strain contains two or even three different toxin genes 32,35,36. In these cases, one toxin is usually expressed at higher levels (designed with a capital letter) than the other toxin (designated with a lower case letter). For instance, strains that express high levels of BoNT/B and low levels of BoNT/F are known as BoNT/Bf strains. There are also cases that one toxin is expressed, but the other toxin is not expressed, which is known as silent toxin (usually marked with ( )). For instance, a survey for infant botulism cases in California showed that 8% strains were BoNT/A(B), which means these strains contain genes for both BoNT/A and BoNT/B, but only express detectable levels of BoNT/A37-39.
As illustrated in the drawings and examples of the present disclosure, published clostridium bacteria genomic sequence databases were searched, and a novel BoNT gene (hereafter designated “BoNT/X”) encoded on the chromosome of Clostridium botulinum strain 111 was identified. Strain 111 was first isolated from an infant botulism patient in Japan in 199640. It has been shown that toxicity from strain 111 in mice can be neutralized by BoNT/B antisera40. It was later confirmed that this strain expresses a subtype of BoNT/B, BoNT/B2, encoded on a plasmid41,42. The sequence of BoNT/X was deposited into PubMed database in February of 2015, as part of genomic sequence of Strain 111. BoNT/X has not been characterized before. It remains unknown whether it is expressed in the strain 111 and whether it is a functional toxin.
Also provided herein are the characterization of BoNT/X at functional levels. Its LC was found to cleave VAMP at a site distinct from known target sites of all other BoNTs. The full-length toxin, produced by covalently linking non-toxic fragments via sortase, was found to enter cultured neurons and cleave VAMP in neurons, inducing flaccid paralysis in mice. Finally, it was found that the toxin is not recognized by antisera raised against all seven known BoNTs, establishing BoNT/X as a novel BoNT serotype. Its identification poses an urgent challenge for developing effective countermeasures. It also has the potential to be developed into a new therapeutic toxin and can be used to generate chimeric toxins with potentially distinct pharmacological properties.
As used herein, the term “Clostridial Botulinum neurotoxin (BoNT) polypeptide” encompasses any polypeptide or fragment from a Botulinum neurotoxin described herein. In some embodiments, the term BoNT refers to a full-length BoNT. In some embodiments, the term BoNT refers to a fragment of the BoNT that can execute the overall cellular mechanism whereby a BoNT enters a neuron and inhibits neurotransmitter release. In some embodiments, the term BoNT simply refers to a fragment of the BoNT, without requiring the fragment to have any specific function or activity. For example, a BoNT polypeptide may refer to the light chain (LC) of a BoNT, e.g., BoNT/X. Other terms that may be used throughout the present disclosure for “Clostridial Botulinum neurotoxins” may be BoNTs, Botulinum toxins, or C. Botulinum toxins. It is to be understood that these terms are used interchangeably. “BoNT/X” refers to the novel BoNT serotype described and characterized in the present disclosure. The BoNT/X protein sequence (GenBank No. BAQ12790.1; four cysteines are underlined and bolded) is also provided:
A “modified Clostridial Botulinum neurotoxin (BoNT)” encompasses a BoNT comprising any modifications in the amino acid sequence, e.g., truncation, addition, amino acid substitution, and any combination thereof. For example, a BoNT/X comprising amino acid substitution mutations in C461 or C467 is a modified BoNT. In another example, a fragment or a domain of the full-length BoNT (e.g., the protease domain, or LC) is considered a modified BoNT. In some embodiments, a domain of the BoNT may also comprise amino acid substitution mutations, e.g., a protease domain comprising substitution mutations at positions C461 or C467 of BoNT/X.
The term “enters a cell” when used to describe the action of a BoNT of the present disclosure, encompasses the binding of a BoNT to a low or high affinity receptor complex, binding of a BoNT to ganglioside, the internalization of the toxin, the translocation of the toxin light chain into the cytoplasm and the enzymatic modification of a BoNT substrate.
As used herein, the term “Clostridial Botulinum neurotoxin (BoNT) protease domain” is synonymous to “light-chain (LC).” The BoNT protease domain is located in the light chain of the BoNT, and thus is also referred to as the LC. The term means a BoNT domain that can execute the enzymatic target modification step of the intoxication process. If the LC from a specific BoNT serotype is referred to, the term “serotype-LC” is used. For example, “X-LC” means the LC polypeptide from BoNT/X. A BoNT protease domain specifically targets a C. Botulinum toxin substrate and encompasses the proteolytic cleavage of a C. Botulinum toxin substrate, such as, e.g., SNARE proteins such as a SNAP-25 substrate, a VAMP substrate and a Syntaxin substrate. In BoNT (e.g., BoNT/X, BoNT/A, BoNT/B, BoNT/C, etc.). The protease domain or the LC is considered to correspond to about amino acid 1-439 of BoNT/X. The domain boundary may vary by about 25 amino acids. For example, the protease domain may correspond to amino acids 1-414 or 1-464 of BoNT/X. In some embodiments, the protease domain may correspond to amino acids 1-438, 1-437, 1-436, 1-435, 1-434, 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 of BoNT/X.
As used herein, the term “Clostridial Botulinum neurotoxin (BoNT) translocation domain“is synonymous with” HN domain” and means a BoNT domain that can execute the translocation step of the intoxication process that mediates BoNT light chain translocation. Thus, an HN facilitates the movement of a BoNT light chain across a membrane into the cytoplasm of a cell. Non-limiting examples of a HN include a BoNT/A HN, a BoNT/B HN, a BoNT/C1 HN, a BoNT/D HN, a BoNT/E HN, a BoNT/F HN, a BoNT/G HN, and a BoNT/X HN. The translocation domain is located in the N-terminus of the heavy chain (HC), and thus is also referred as HN. It is to be understood that these terms are used interchangeably herein.
As used herein, the term “linker region” refers to the amino acid sequence between the BoNT protease domain and the translocation domain. The linker comprises two cysteines at position 461 and 467, one of which forms an inter-molecular disulfide bond with a cysteine in the protease domain, C423 (C461-C423 disulfide bond, or C467-C423 disulfide bond). The formation of this disulfide bond is essential for the activity of BoNT/X.
As used herein, the term “LC-HN” refers to a BoNT polypeptide encompassing the protease domain, the linker region, and the translocation domain. If the LC-HN from a specific BoNT serotype is referred to, the term “serotype-LC-HN” is used. For example, “X-LC-HN” means the LC-HN polypeptide from BoNT/X. The LC-HN polypeptide is considered to correspond to about amino acid 1-892 of BoNT/X. The domain boundary may vary by about amino acids. For example, LC-HN polypeptide may correspond to about amino acid 1-917 or 1-867 of BoNT/X. In some embodiments, the LC-HN 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 of BoNT/X.
As used herein, the term “Clostridial Botulinum neurotoxin (BoNT) receptor-binding domain” is synonymous with “HC domain” and means any naturally occurring BoNT receptor binding domain that can execute the cell binding step of the intoxication process, including, e.g., the binding of the BoNT to a BoNT-specific receptor system located on the plasma membrane surface of a target cell. Some aspects of present disclosure relate to modified BoNT receptor binding domains from serotype X (BoNT/X). In some embodiments, a “modified BoNT/X receptor binding domain” comprises amino acid substitutions in a position corresponding to C1240 in BoNT/X (SEQ ID NO: 1). The receptor binding domain, or the HC, is considered to correspond to about amino acid 893-1306 of BoNT/X. The domain boundary may vary by about 25 amino acids. For example, the receptor binding domain or HC may correspond to amino acids 868-1306 or 918-1306. In some embodiments, the receptor binding domain or HC 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 of BoNT/X.
By “isolated” is meant a material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings, e.g., from a cell or from flanking DNA or from the natural source of the DNA. The term “purified” is used to refer to a substance such as a polypeptide that is “substantially pure”, with respect to other components of a preparation (e.g., other polypeptides). It can 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, with respect to other components. The terms “substantially pure” or “essentially purified”, with regard to a polypeptide, refers to a preparation that contains fewer than about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of one or more other components (e.g., other polypeptides or cellular components).
The term “substitution mutation” without the reference to a specific amino acid, may include any amino acid other than the wild type residue normally found at that position. Such substitutions may be replacement with non-polar (hydrophobic) amino acids, such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Substitutions may be replacement with polar (hydrophilic) amino acids such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Substitutions may be replacement with electrically charged amino acids, e.g., 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 typically be replacement with a different naturally occurring amino acid residue, but in some cases non-naturally occurring amino acid residues may also be substituted. Non-natural amino acids, as the term is used herein, are non-proteinogenic (i.e., non-protein coding) amino acids that either occur naturally or are chemically synthesized. Examples include but are not limited to β-amino acids (β and β2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methyl amino acids. In some embodiments, the amino acid can be substituted or unsubstituted. The substituted amino acid or substituent can be a halogenated aromatic or aliphatic amino acid, a halogenated aliphatic or aromatic modification on the hydrophobic side chain, or an aliphatic or aromatic modification.
The “percent identity” of 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 incorporated into the NBLAST and XBLAST programs (version 2.0) of 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 the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Accordingly, some aspects of the present disclosure provide isolated BoNT polypeptides. In some embodiments, the isolated BoNT polypeptide is a full-length BoNT/X polypeptide. In some embodiments, the isolated BoNT polypeptide comprise the a amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated BoNT/X polypeptide comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 1. For example, the isolated BoNT polypeptide may comprise an amino acid sequence that has 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 to SEQ ID NO: 1. In some embodiments, the isolated BoNT polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89% 0.90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 1. In some 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-HN polypeptide. In some embodiments, the isolated BoNT polypeptide comprise the a amino acid sequence of SEQ ID NO: 2. In some embodiments, the isolated BoNT polypeptide comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 2. For example, the isolated BoNT polypeptide may comprise an amino acid sequence that has 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 to SEQ ID NO: 2. In some embodiments, the isolated BoNT polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89% 0.90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 2. 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 comprise the a amino acid sequence of SEQ ID NO: 3. In some embodiments, the isolated BoNT polypeptide comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 3. For example, the isolated BoNT polypeptide may comprise an amino acid sequence that has 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 to SEQ ID NO: 3. In some embodiments, the isolated BoNT polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 3. In some embodiments, the isolated BoNT polypeptide consists of the amino acid sequence of SEQ ID NO: 3.
The X-LC polypeptide may be introduced alone into cells where the cleavage of a BoNT substrate (e.g., a SNARE protein) is desired for research or therapeutic purpose, by any known techniques of expression an exogenous protein in the art, e.g., transfection of LC coding sequence directly into cells, via lentiviral vectors, via AAV vectors, or fusing X-LC with cell penetrating peptides).
In some embodiments, the BoNT polypeptides of the present disclosure is a full-length BoNT/X comprising a protease domain (LC), a linker region, a translocation domain (HN), and a receptor binding domain (HC), wherein the linker region 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 via the cleavage of the linker region between LC and HN either bacterial or host proteases. This process is known as “activation” and is essential for the activity of BoNT/X After the cleavage, the LC and HN remain connected via an inter-chain disulfide bond prior to translocation of LC into the cytosol of cells, where the disulfide bond is reduced in order 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), which is unique to BoNT/X. The formation of the inter-chain disulfide bond (C423-C461, or C423-C467) is required for BoNT/X activity.
In addition to the cysteines in the linker region, the receptor binding domain of BoNT contains another cysteine, C1240, which can also form inter-molecular disulfide bonds with other cysteines in BoNT/X. These intermolecular disulfide bonds causes BoNT/X to aggregate and destabilizes the protein (
Accordingly, some aspects of the present disclosure provide modified BoNT/X polypeptide comprising one or more substitution mutation(s) in C461, C467, or C1240, which are more stable than the wild-type BoNT/X and have comparable activities. The cysteines may be substituted with any amino acids that abolish the formation of disulfide bonds. In some embodiments, the cysteines are substituted with serine (S) or alanine (A). Possible combinations of substitution mutations that may be present in the modified BoNTs of the present disclosure are, without limitation: C461S, C461A, C467S, C467A, C1240S, C1240A, C461S/C1240S, C461A/C1240S, C461S/C1240A, C467A/C1240A, C467S/C1240S, C467A/C1240S, C467S/C1240A, and C467A/C1240A. “/” indicates double mutations. In some embodiments, the modified BoNT/X polypeptide of the present disclosure comprises an amino acid sequence of any one of SEQ ID NOs: 4-17. In some embodiments, the modified BoNT/X polypeptide comprises an amino acid sequence that has at least 85% identity to any one of SEQ ID NO: 4-17, and does not have the amino acid sequence of SEQ ID NO: 1. For example, the modified BoNT/X polypeptide may comprise an amino acid sequence that has 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 to any one of SEQ ID NOs: 4-17, and does not have the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified BoNT/X polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 4-17, and does not have the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified BoNT/X polypeptide 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-HN polypeptide comprising the substitution mutations described herein. In some embodiments, the modified BoNT/X-LC-HN comprises one single substitution mutation in a position corresponding to C461 or C467 in SEQ ID NO: 2. In some embodiments, the modified BoNT/X-LC-HN comprises 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 an amino acid sequence of any one of SEQ ID NOs: 18-21. In some embodiments, the modified BoNT/X-LC-HN polypeptide comprises an amino acid sequence that has at least 85% identity to any one of SEQ ID NO: 18-21, and does not have the amino acid sequence of SEQ ID NO: 2. For example, the modified BoNT/X-LC-HN polypeptide may comprise an amino acid sequence that has 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 to any one of SEQ ID NOs: 18-21, and does not have the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified BoNT/X-LC-HN polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 18-21, and does not have the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified BoNT/X-LC-HN polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 18-21.
The modified BoNT polypeptide comprising one or more substitution mutation(s) (e.g., in C461, C467, or C1240) described herein does not form inter-molecular disulfide bonds that cause aggregation of the protein, and are therefore more stable than their corresponding wild type proteins. The activity of the BoNT polypeptides are not affected by the substitution mutations in the cysteines. Thus, the modified BoNT/X may be more suitable for therapeutic use than the wild type BoNT/X due to its increased stability.
Other aspects of the present disclosure provide chimeric BoNTs comprising BoNT/X-LC-HN described herein and the receptor binding domain (HC) from a different BoNT. For example, the receptor binding domain may be from any one of BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/E, BoNT/F, and BoNT/G. Thus, the chimeric BoNTs contemplated herein include BoNT/X-LC-HN-A-HC, BoNT/X-LC-HN-B-HC, BoNT/X-LC-HN-C-HC, BoNT/X-LC-HN-D-HC, BoNT/X-LC-HN-E-HC, BoNT/X-LC-HN-F-HC, and BoNT/X-LC-HN-G-HC. It is to be understood that the HC domain of any subtypes of the seven known scrotypes (e.g., A, B, C, D, E, F, or G) are suitable for the chimeric toxin. When BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, or BoNT/G is referred to, it encompasses all the subtypes. For example, BoNT/A has 8 subtypes, BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, BoNT/A5, BoNT/A6, BoNT/A7, or BoNT/A8, and the HC of any one of these BoNT/A subtypes are suitable for use in the chimeric BoNT of the present disclosure. Similarly, the HC of any one of the 8 subtypes of BoNT/B, i.e., BoNT/B1, BoNT/B2, BoNT/B3, BoNT/B4, BoNT/B5, BoNT/B6, BoNT/B7, or BoNT/B8, are suitable for use in the chimeric BoNT of the present disclosure.
In some embodiments, BoNT/X-LC-HN-A1-HC (SEQ ID NO: 22), BoNT/X-LC-HN-B1-HC (SEQ ID NO: 23), and BoNT/X-LC-HN-C1-HC (SEQ ID NO: 24) are provided. In some embodiments, the chimeric BoNT polypeptide comprises an amino acid sequence that has at least 85% identity to any one of SEQ ID NO: 22-24. For example, the chimeric BoNT polypeptide may comprise an amino acid sequence that has 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 to any one of SEQ ID NOs: 22-24. In some embodiments, the chimeric BoNT polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 22-24. In 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 of the present disclosure comprises a modified BoNT/X-LC-HN comprising a substitution mutation in the linker region, e.g., in a position corresponding to C461 or C467 of SEQ ID NO: 2. For example, the BoNT/X-LC-HN in the chimeric BoNT may comprise a substitution mutation corresponding to C461A, C467A, C461S, or C467S of SEQ ID NO: 2. For example, the chimeric BoNT polypeptide of the present disclosure may comprise an 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 has at least 85% identity to any one of SEQ ID NO: 25-30. For example, the chimeric BoNT polypeptide may comprise an amino acid sequence that has 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 to any one of SEQ ID NOs: 25-30. In some embodiments, the chimeric BoNT polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 25-30. In some embodiments, the chimeric BoNT polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 25-30.
To generate the chimeric toxins, e.g., the BoNT/X-LC-HN-A1-HC toxin, the X-LC-HN fragment comprising amino acid of about 1-892 (SEQ ID NO: 2) is fused to the receptor binding domain of any one of BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/E, BoNT/F, and BoNT/G. The receptor binding domains of different BoNTs correspond to amino acids of about 860-1291 of BoNT/B1. It is to be understood that the border of the X-LC-HN fragment and/or the receptor binding domains may vary by 1-25 amino acids. For example, the X-LC-HN fragment that may be used for the chimeric toxin may comprise amino acids 1-917 or 1-867 of BoNT/X. In some embodiments, the X-LC-HN fragment that may be used for the chimeric toxin may comprise 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 of BoNT/X. Similarly, the receptor binding that may be used for the chimeric toxin may comprise amino acid corresponding to 885-1291 or 835-1291 of BoNT/X. In some embodiments, the receptor binding that may be used for the chimeric toxin may comprise amino acid corresponding to 860-1291, 861-1291, 862-1291, 863-1291, 864-1291, 865-1291, 866-1291, 867-1291, 868-1291, 869-1291, 870-1291, 860-1291, 859-1291, 858-1291, 857-1291, 856-1291, 855-1291, 854-1291, 853-1291, 852-1291, or 851-1291 of BoNT/B. The skilled artisan is able to identified the domains that may be used for the chimeric toxin of the present disclosure, based on his/her knowledge in protein homology, with or without the assistance of a sequence alignment software. The methods of fusing the fragments are standard recombinant techniques that are well known to one skilled in the art.
Further contemplated herein are modified BoNT/X polypeptides comprising a modified linker region, wherein the linker region comprises a specific protease cleavage site. A “specific protease cleavage site,” as used herein, refers to a recognition and cleavage site for a specification protease, as opposed to a sequence that is recognized and cleavage by more than one non-specific proteases. Such specific proteases include, without limitation: thrombin, TEV, PreScission, Factor Xa, MMP-12, MMP-13, MMP-17, MMP-20, Granzyme-B, and Enterokinase. The cleavage site of the specific proteases may be added to the linker region of the BoNT/X polypeptide via insertion or replacement of the existing amino acids in the linker region (e.g., replace amino acids 424-460 of the BoNT/X polypeptide). The sequences of the specific protease cleavage sites sequences are also provided: 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). “|” indicates where cleavage occurs.
Other aspects of the present disclosure provide the functional characterization of the BoNT/X polypeptides. The BoNT/X polypeptides, modified BoNT/X polypeptides, and chimeric BoNT polypeptides of the present disclosure can bind and enter target cells, e.g., neurons, and cleave its substrate proteins, e.g. SNARE proteins. The term “SNARE proteins,” as used herein, refers to SNAP (Soluble NSF Attachment Protein) Receptors, which is a large protein superfamily consisting of more than 60 members in yeast and mammalian cells. The primary role of SNARE proteins is to mediate vesicle fusion, i.e., the fusion of vesicles with their target membrane bound compartments (such as a lysosome). The best studied SNARE proteins are those that mediate docking of synaptic vesicles with the presynaptic membrane in neurons, e.g., SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, VAMP7, VAMP8, syntaxin1, and Ykt6. Several of these SNARE proteins are substrates of BoNTs. For example, VAMP1, VAMP2, VAMP3, SNAP-25, and syntaxin 1 have been shown to be cleaved by known BoNTs, e.g., BoNT/A and BoNT/B.
Provided herein are data showing that BoNT/X cleaves the SNARE proteins that are known substrates of BoNTs. One surprising finding of the present disclosure is that BoNT/X is able to cleave several SNARE proteins that other BoNTs are not able to cleave, e.g., VAMP4, VAMP5, and Ykt6. VAMP4 is widely expressed and is known to mediate vesicle fusion between trans-Golgi network (TGN) and endosomes, as well as homotypic fusion of endosomes. BoNTs are traditionally known to be limited to target SNAREs that mediate vesicle exocytosis onto plasma membranes. BoNT/X is the first BoNT that is capable of cleaving SNAREs mediating other type fusion events inside cells that is not with plasma membrane as the destine. VAMP4 may also contribute to asynchronous synaptic vesicle exocytosis, enlargeosome exocytosis, and activity-dependent bulk endocytosis (ADBE) in neurons. In addition, VAMP4 has been implicated in granule release in immune cells. Thus, BoNT/X might have a unique potential among all BoNTs to modulate inflammatory secretion in immune cells, which can be exploited therapeutically. VAMP5 is mainly expressed in muscles and its function remains to be established. BoNT/X will be a unique tool for investigating the function of VAMP4 and VAMP5. Ykt6 functions in endoplasmic reticulum to Golgi transport. It also functions in early/recycling endosome to TGN transport. The identification of Ykt6 as a substrate of the BoNT polypeptides described herein is significant because it opens up new therapeutic application for blocking secretion in a wide range of cells by BoNTs.
Another surprising finding of the present disclosure is that BoNT/X cleaves the SNARE proteins at a novel site what was not previously described. As illustrated in the Examples and Figures of the present disclosure, BoNT/X cleaves between amino acids R66-S67 in VAMP1, VAMP2, and VAMP3. R66-A67 is a novel cleavage site distinct from established target sites for all other BoNTs (
Accordingly, the BoNT polypeptides of the present disclosure have expanded profile of target cells and substrates. In some embodiments, the BoNT polypeptide cleaves a 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 amino acid residues corresponding to R66 and A67 of 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 amino acid residues corresponding to R66 and A67 of 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 amino acid residues corresponding to R66 and A67 of 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 amino acid residues corresponding to K87 and S88 of 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 amino acid residues corresponding to R40 and S41 of 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 amino acid residues corresponding to K173 and S174 of SEQ ID NO: 44.
In some embodiments, the BoNT polypeptide of the present disclosure cleaves a SNARE protein in a target cell. As used herein, a “target cell” means a cell that is a naturally occurring cell that BoNT is capable of entering or intoxicating. In some embodiments, a target cell is a secretory cell, e.g., a neuron or a secretory immune cell. Examples of neurons that may be BoNT target cells include, without limitation, motor neurons; sensory neurons; autonomic neurons; such as, e.g., sympathetic neurons and parasympathetic neurons; non-peptidergic neurons, such as, e.g., cholinergic neurons, adrenergic neurons, noradrenergic neurons, serotonergic neurons, GABAergic neurons; and peptidergic neurons, such as, e.g., Substance P neurons, Calcitonin Gene Related Peptide neurons, vasoactive intestinal peptide neurons, Neuropeptide Y neurons, cholecystokinin neurons.
The BoNT polypeptide of the present disclosure, e.g., the BoNT/X or the modified BoNT/X polypeptide, is able to target other types of secretory cells 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. A “secretory immune cell,” as used herein, refers to immune cells that secrets cytokines, chemokines, or antibodies. Such secretory immune cells may be innate immune cells including, without limitation, natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. Secretory immune cells that secret antibodies (e.g., white blood cells) may also be targeted by the BoNT polypeptides of the present disclosure. Non-limiting examples of antibody secreting cells include, without limitation, plasma B cells, plasmocytes, plasmacytes, and effector B cells. In some embodiments, the target cell is a cultured cell, e.g., a cultured neuron or a cultured secretory immune cell. In some embodiments, the target cell is in vivo. In some embodiments, target cell is from a mammal. In some embodiments, the mammal is a human. In embodiments, the mammal is a rodent, e.g., a mouse or a rat.
In some embodiments, the BoNT polypeptide suppresses neuronal activity. In some embodiments, the BoNT polypeptide modulates immune response. In some embodiments, the BoNT polypeptide induces flaccid paralysis. “Flaccid paralysis” refers to a clinical manifestation characterized by weakness or paralysis and reduced muscle tone without other obvious cause (e.g., trauma).
Other aspects of the present disclosure provide modified BoNT/X polypeptides comprising an inactive protease domain. Such BoNT/X polypeptides (also referred to herein as “inactive BoNT/X”) can enter the target cells but cannot cleave the substrate proteins (e.g., a SNARE protein) due to the inactivation of the protease domain. In some embodiments, the inactive BoNT/X is an X-LC-HN fragment comprising: a) an inactive protease domain; b) a linker region; 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) a linker region; c) a translocation domain; and d) a receptor binding domain. In some embodiments, the inactive protease domain comprises one or more substitution mutation(s) in a position corresponding to R360, Y363, H227, E228, or H231 of SEQ ID NO: 1. In some embodiments, the one or more substitution mutation(s) corresponds to R360A/Y363F, H227Y, E228Q, or H231Y in SEQ ID NO: 1. It is to be understood that the inactive BoNT/X polypeptide may comprise any mutation(s) that inactivates the protease domain.
In some embodiments, the inactive BoNT/X polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 31-38. In some embodiments, the inactive BoNT/X polypeptide comprises an amino acid sequence that has at least 85% identity to any one of SEQ ID NOs: 31-38. For example, the inactive BoNT/X polypeptide may comprise an amino acid sequence that has 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 to any one of SEQ ID NOs: 31-38. In some embodiments, the inactive BoNT/X polypeptide comprises an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of SEQ ID NOs: 31-38. In some embodiments, the inactive BoNT/X polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 31-38.
In some embodiments, the inactive BoNT/X (e.g., inactive X-LC-HN or inactive full length BoNT/X) further comprises mutations in the linker region. In some embodiments, the modification in the linker region comprises one single substitution mutation in a position corresponding to C461 or C467 of SEQ ID NO: 1. In some embodiments, the single substitution mutation corresponds to C461A, C461S, C467A, or C467S in SEQ ID NO: 1. In some embodiments, the inactive BoNT/X (e.g., the 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 in a position corresponding to C1240 of SEQ ID NO: 1.
It is also envisioned that the modified BoNT/X polypeptide comprising an inactive protease domain described herein can be utilized as a delivery tool to target cells (e.g., neurons) in humans. For example, the modified BoNT/X can be linked to other therapeutic agents, covalently or non-covalently, and acts as the targeting vehicle to deliver the therapeutic agents to target cells in humans.
As such, another aspect of the disclosure relates to a chimeric polypeptide molecule comprising a first portion that is an inactive BoNT/X, comprising one or more substitution mutations that inactivates the protease domain, linked to a second portion. The second portion of the molecule can be a bioactive molecule such as a therapeutic agent (e.g., a polypeptide or non-polypeptide drug). Linkage of the first and second portions of the molecule can be covalent (e.g., in the form of a fusion protein) or non-covalent. Methods of such linkage are known in the art and can readily be applied by the skilled practitioner. When the second portion of the chimeric molecule is a polypeptide and the chimeric molecule is in the form of a protein, nucleic acids and nucleic acid vectors encoding such chimeric molecules are provided.
Also provided are cells comprising the nucleic acids or nucleic acid vectors, and cells expressing such chimeric molecules. The chimeric molecules in a fusion protein form may be expressed and isolated using the methods disclosed herein.
The modified BoNT/X polypeptides, the chimeric BoNT polypeptides, or the chimeric molecules comprising a second portion that is a polypeptide of the present disclosure (e.g., without limitation, polypeptides comprising amino acid sequence of any one of SEQ ID NOs: 1-38), will generally be produced by expression form recombinant nucleic acids in appropriate cells (e.g., E. coli, or insect cells) and isolated. The nucleic acids encoding the polypeptides described herein may be obtained, and the nucleotide sequence of the nucleic acids determined, by any method known in the art.
Further provided herein are isolated and/or recombinant nucleic acids encoding any of the BoNT polypeptides disclosed herein. The nucleic acids encoding the isolated polypeptide fragments of the present disclosure, may be DNA or RNA, double-stranded or single stranded. In certain aspects, the subject nucleic acids encoding the isolated polypeptide fragments are further understood to include nucleic acids encoding polypeptides that are variants of any one 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, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 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 of any one of SEQ ID NOs: 1-38. In some embodiments, the isolated nucleic acid molecule of the present disclosure comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity of any one of SEQ ID NOs: 1-38.
In some embodiments, the nucleic acid is comprised within a vector, such as an expression vector. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid.
A variety of promoters can be used for expression of the polypeptides described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter. Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those 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, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad. Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [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 the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (HCMV) major immediate-early promoter to create a 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)).
Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and 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 comprising the nucleic acid can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the polypeptides described herein. In some embodiments, the expression of the polypeptides described herein is regulated by a constitutive, an inducible or a tissue-specific promoter.
The host cells used to express the isolated polypeptides described herein may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells. In particular, mammalian cells, such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al. (1986) “Powerful And Versatile Enhancer-Promoter Unit For Mammalian Expression Vectors,” Gene 45:101-106; 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 variety of host-expression vector systems may be utilized to express the isolated polypeptides described herein. Such host-expression systems represent vehicles by which the coding sequences of the isolate d polypeptides described herein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the isolated polypeptides described herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., 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 (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the isolated polypeptides described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) containing the sequences encoding the isolated polypeptides described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the isolated polypeptides described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (human retinal cells developed by Crucell) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the polypeptides being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of polypeptides described herein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (RUther et al. (1983) “Easy Identification Of cDNA Clones,” EMBO J. 2:1791-1794), in which the coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; 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 may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (e.g., see 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 initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al. (1987) “Expression And Secretion Vectors For Yeast,” Methods in Enzymol. 153:516-544). In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. For example, in certain 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 mechanisms to form separate polypeptides described herein.
The disclosure thus encompasses engineering a nucleic acid sequence to encode 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. The post translational cleavage of the precursor molecule comprising the polypeptides described herein may occur in vivo (i.e., within the host cell by native or recombinant cell systems/mechanisms, e.g. furin cleavage at an appropriate site) or may occur in vitro (e.g. incubation of said polypeptide chain in a composition comprising proteases or peptidases of known activity and/or in a composition comprising conditions or reagents known to foster the desired proteolytic action).
Purification and modification of recombinant proteins is well known in the art such that the design of the polyprotein precursor could include a number of embodiments readily appreciated by a skilled worker. Any known proteases or peptidases known in the art can be used for the described modification of the precursor molecule, e.g., 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 by reference herein 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 herein in its entirety)), furin, and AcTEV (Parks et al. (1994) “Release Of Proteins And Peptides From Fusion Proteins Using A Recombinant Plant Virus Proteinase,” Anal. Biochem. 216:413-417 hereby incorporated by reference herein in its entirety)) and the Foot and Mouth Disease Virus Protease C3.
Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express polypeptides described herein may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the polypeptides described herein. Such engineered cell lines may be particularly useful in screening and evaluation of polypeptides that interact directly or indirectly with the polypeptides described herein.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) “Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Cultured Mouse Cells,” Cell 11: 223-232), hypoxanthine-guanine phosphoribosyltransferase (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 phosphoribosyltransferase (Lowy et al. (1980) “Isolation Of Transforming DNA: Cloning The Hamster aprt Gene,” Cell 22: 817-823) genes can be employed in tk−, hgprt− or aprt− cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, 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 confers 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, which confers resistance to the aminoglycoside 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. 62:191-217) and hygro, which confers resistance to hygromycin (Santerre et al. (1984) “Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L Cells,” Gene 30:147-156). Methods commonly known in the art of recombinant DNA tecHNology 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.
The expression levels of 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). When a marker in the vector system expressing a polypeptide described herein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a polypeptide described herein or a polypeptide described herein, production of the polypeptide will also increase (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 may be purified by any method known in the art for purification of polypeptides, polyproteins or antibodies (e.g., analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the polypeptide comprises an Fe domain (or portion thereof)), and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies. Other aspects of the present disclosure relate to a cell comprising a nucleic acid described herein or a vector described herein.
The cell may be a prokaryotic or eukaryotic cell. In some embodiments, the cell in a mammalian cell. Exemplary cell types are described herein. Other aspects of the present disclosure related to a cell expressing the modified BoNT polypeptides described herein. The cell may be a prokaryotic or eukaryotic cell. In some embodiments, the cell in a mammalian cell. Exemplary cell types are described herein. The cell can be for propagation of the nucleic acid or for expression of the nucleic acid, or both. Such cells include, without limitation, prokaryotic cells including, without limitation, strains of aerobic, microaerophilic, capnophilic, facultative, anaerobic, gram-negative and gram-positive bacterial cells such as those derived from, e.g., 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, without limitation, yeast strains, such as, e.g., 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, e.g., those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca sexta; and mammalian cells and cell lines derived from mammalian cells, such as, e.g., those derived from mouse, rat, hamster, porcine, bovine, equine, primate and human. Cell lines may be obtained from the American Type Culture Collection, European Collection of Cell Cultures and the German Collection of Microorganisms and Cell Cultures. Non-limiting examples of specific protocols for selecting, making and using an appropriate cell line are described in e.g., 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 one skilled in the art and from the teaching herein. Yet other aspects of the present disclosure relate to a method of producing a polypeptide described herein, the method comprising obtaining a cell described herein and expressing nucleic acid described herein in said cell. In some embodiments, the method further comprises isolating and purifying a polypeptide described herein.
In some embodiments, botulinum neurotoxin can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive.
The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C, D and E are synthesized by non-proteolytic strains and are therefore typically inactive when recovered from culture. Serotypes 13 and F are produced by both proteolytic and non-proteolytic strains and therefore can be recovered in either the active or inactive form. The proteolytic strains that produce, for example, the botulinum toxin type B serotype may only cleave a portion of the toxin produced. The production of BoNT/X polypeptides using these strains are contemplated herein.
The exact proportion of nicked to un-nicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of a preparation of, for example, the botulinum toxin type B toxin 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 envisioned.
One aspect of the present disclosure provides novel methods of producing BoNTs via an in vitro transpeptidase reaction that ligates two non-toxic fragments of BoNTs. Such methods comprise the steps of: (i) obtaining a first BoNT fragment comprising a light chain (LC) and a N-terminal domain of a heavy chain (HN), wherein the first BoNT fragment comprises a C-terminal LPXTGG (SEQ ID NO: 60) motif; (ii) obtaining a second BoNT fragment comprising a C-terminal domain of the heavy chain (HC); wherein the second BoNT fragment comprise a specific protease cleavage site at its N-terminus; (iii) cleaving 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 the X-LC-HN polypeptide described herein fused to a C-terminal LPXTGG (SEQ ID NO: 60) motif (e.g., SEQ ID NO: 45), or any variants thereof. In some embodiments, the second BoNT fragment comprises the He polypeptide described herein, or any variants thereof (e.g., SEQ ID NO: 46). It is to be understood that any BoNT fragments or domains may be ligated using the methods described herein.
The methods described herein may 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. One skilled in the art will be able to discern the combinations that may be made. In some embodiments, the chimeric BoNT polypeptides described herein (e.g., BoNT/X-LC-HN-A1-HC, BoNT/X-LC-HN-B1-HC, or BoNT/X-LC-HN-C1-HC) are made using this method.
In some embodiments, the transpeptidase is a sortase. In some embodiments, the sortase is from Staphylococcus aureus (SrtA).
Other peptide ligation systems available in the art may also be used to ligate two non-toxic BoNT fragments. For example, an intein-mediated protein ligation reaction allows the ligation of a synthetic peptide or a protein with an N-terminal cysteine residue to the C-terminus of a bacterially expressed protein through 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. USA92, 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 references). Kits are commercially available (e.g., from New England Biolabs) for intern-mediated protein ligation reactions.
In some embodiments, the first BoNT fragment further comprises an affinity tag. In some embodiments, the affinity tag is fused to first BoNT fragment at the N-terminus. In some embodiments, the affinity tag is fused to the first BoNT fragment at the C-terminus. In the event that the affinity tag is fused to the C-terminus of the first BoNT fragment, the transpeptidase cleaves between the T and G in the LPXTGG (SEQ ID NO: 60) motif and removes the affinity tag before ligating the first BoNT fragment and the second BoNT fragment.
In some embodiments, the second BoNT fragment further comprises an affinity tag. In 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 the second BoNT fragment at the C-terminus. In the event that the affinity tag is fused to the N-terminus of the first BoNT fragment, the specific protease cleaves in the specific protease cleavage site and removes the affinity tag before ligating the first BoNT fragment and the second BoNT fragment by the transpeptidase.
An “affinity tag,” as used herein, refers to a polypeptide sequence that can bind specifically to a substance or a moiety, e.g., a tag comprising six Histidines bind specifically to Ni2+. Affinity tags may be appended to proteins to facilitate their isolation. The affinity tags are typically fused to proteins via recombinant DNA techniques known by those skilled in the art. The use of affinity tags to facilitate protein isolate is also well known in the art. Suitable affinity tags that may 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 Fe.
The second BoNT fragment has a specific protease cleavage at the N-terminus. Cleavage of the site by the specific protease results to a free glycine residue at the N-terminus of the second BoNT fragment. Suitable specific protease that may be used in accordance with the present disclosure include, without limitation: thrombin, TEV, PreScission, Enterokinase, and SUMO protease. In some embodiments, the specific protease is thrombin, and the cleavage site is: LVPRIGS (SEQ ID NO: 50).
The BoNT/X polypeptides described herein affords potential for therapeutic use. For example, BoNT/X might be more potent compared to other BoNT serotypes. BoNT/X is more versatile 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 contemplates pharmaceutically compositions comprising the BoNT/X polypeptides or the chimeric molecules of the present disclosure. As it may also become clear later in the present disclosure, the pharmaceutical composition of the present disclosure, may further comprise other therapeutic agents suitable for the specific disease such composition is designed to treat. In some embodiments, the pharmaceutically composition of the present disclosure further comprises pharmaceutically-acceptable carriers.
The term “pharmaceutically-acceptable carrier”, as used herein, means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the polypeptide from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which 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, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, 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) polyols, such as glycerin, 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 water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, a BoNT polypeptide of the present disclosure in a composition is administered by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
Typically, when administering the composition, materials to which the polypeptide of the disclosure does not absorb are used. In other embodiments, the BoNT polypeptides of the present disclosure are delivered in a controlled release system. Such compositions and methods for administration are provides in U.S. Patent publication No. 2007/0020295, the contents of which are herein incorporated by reference. In one embodiment, a pump may be used (see, e.g., 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). In another embodiment, polymeric materials can be used. (See, e.g., 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, for example, in Langer, supra.
The BoNT polypeptides of the present disclosure can be administered as pharmaceutical compositions comprising a therapeutically effective amount of a binding agent and one or more pharmaceutically compatible ingredients. In typical embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human being.
Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein.
The polypeptides of the present disclosure can be entrapped in ‘stabilized plasmid-lipid particles’ (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al, Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 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 reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle. In some embodiments, the BoNT/X polypeptides described herein may be conjugated to a therapeutic moiety, e.g., an antibiotic. TecHNiques for conjugating such therapeutic moieties to polypeptides, including e.g., Fe domains, are well known; see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp. 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, pp. 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, pp. 303-16, Academic Press; and Thorpe et al. (1982) “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,” Immunol. Rev., 62:119-158. Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a polypeptide of the disclosure in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized polypeptide of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label.
Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. 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 of the disclosure. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
The BoNT polypeptides (e.g., BoNT/X polypeptides), the chimeric molecules, and the pharmaceutical compositions of the present disclosure may be used for the treatment of 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 the BoNT polypeptide, the chimeric molecule, or the pharmaceutical composition described herein to thereby treat the condition. In some embodiments, the BoNT polypeptides, the chimeric molecules, and the pharmaceutic compositions of the present disclosure contact one or more neuron(s) exhibiting unwanted neuronal activity,
Conditions typically treated with a neurotoxin (e.g., skeletal muscle conditions, smooth muscle conditions, glandular conditions, a neuromuscular disorder, an autonomic disorder, pain, or an aesthetic/cosmetic condition) are associated with unwanted neuronal activity, as determined by the skilled practitioner. Administration is by a route that contacts an effective amount of the composition to neurons exhibiting the unwanted activity. In some embodiments, the condition may be associated with overactive neurons or glands. Specific conditions envisioned for treatment by the methods discussed herein include, without limitation, spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions as well as other secretory disorders, pain from muscle spasms, headache pain. In addition, the present disclosure can be used to treat dermato logical or aesthetic/cosmetic conditions, for example, reduction of brow furrows, reduction of skin wrinkles.
One unique property of the BoNT/X polypeptides of the present disclosure is its ability to cleave VAMP4, VAMP5, and Ykt6. Thus, further contemplated herein are therapeutic use of the BoNT/X polypeptides in conditions associated with unwanted secretion activities in a wide range of cells. In some embodiments, the unwanted secretion is immune secretion. Conditions associated with unwanted immune secretion include, without limitation: inflammation, psoriasis, allergy, haemophagocytic lymphohistiocytosis, and alcoholic pancreatic disease.
The present disclosure can also be used in the treatment of sports injuries. Borodic U.S. Pat. No. 5,053,005 discloses methods for treating juvenile spinal curvature, i.e. scoliosis, using botulinum type A. The disclosure of Borodic is incorporated in its entirety herein by reference. In one embodiment, using substantially similar methods as disclosed by Borodic, a BoNT polypeptide can be administered to a mammal, preferably a human, to treat spinal curvature. In a suitable embodiment, a BoNT polypeptide comprising botulinum type E fused with a leucine-based motif is administered. Even more preferably, a BoNT polypeptide comprising botulinum type A-E with a leucine-based motif fused to the carboxyl terminal of its light chain is administered to the mammal, preferably a human, to treat spinal curvature.
In addition, the 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, for example, headache pain, pain from muscle spasms and various forms of inflammatory pain. For example, Aoki U.S. Pat. No. 5,721,215 and Aoki U.S. Pat. No. 6,113,915 disclose methods of using botulinum toxin type A for treating pain. The disclosure of these two patents is incorporated in its entirety herein by reference.
Autonomic nervous system disorders can also be treated with a modified neurotoxin. For example, glandular malfunctioning is an autonomic nervous system disorder. Glandular malfunctioning includes excessive sweating and excessive salivation. Respiratory malfunctioning is another example of an autonomic nervous system disorder. Respiratory malfunctioning includes chronic obstructive pulmonary disease and asthma. Sanders et al. disclose methods for treating the autonomic nervous system; for example, treating autonomic nervous system disorders such as excessive sweating, excessive salivation, asthma, etc., using naturally existing botulinum toxins. The disclosure of Sander et al. is incorporated in its entirety by reference herein.
In one embodiment, substantially similar methods to that of Sanders et al. can be employed, but using a BoNT polypeptide, to treat autonomic nervous system disorders such as the ones discussed above. For example, a BoNT polypeptide can be locally applied to the nasal cavity of the mammal in an amount sufficient to degenerate cholinergic neurons of the autonomic nervous system that control the mucous secretion in the nasal cavity. Pain that can be treated by a modified neurotoxin includes pain caused by muscle tension, or spasm, or pain that is not associated with muscle spasm. For example, Binder in U.S. Pat. No. 5,714,468 discloses that headache caused by vascular disturbances, muscular tension, neuralgia and neuropathy can be treated with a naturally occurring botulinum toxin, for example Botulinum type A. The disclosures of Binder are incorporated in its entirety herein by reference.
In one embodiment, substantially similar methods to that of Binder can be employed, but using a BoNT polypeptide described herein, to treat headache, especially the ones caused by vascular disturbances, muscular tension, neuralgia and neuropathy. Pain caused by muscle spasm can also be treated by an administration of a BoNT polypeptide described herein. For example, a botulinum type E fused with a leucine-based motif, preferably at the carboxyl terminal of the botulinum type E light chain, can be administered intramuscularly at the pain/spasm location to alleviate pain. Furthermore, a modified neurotoxin can be administered to a mammal to treat pain that is not associated with a muscular disorder, such as spasm.
In one broad embodiment, methods of the present disclosure to treat non-spasm related pain include central administration or peripheral administration of the BoNT polypeptide. For example, Foster et al. in U.S. Pat. No. 5,989,545 discloses that a botulinum toxin conjugated with a targeting moiety can be administered centrally (intrathecally) to alleviate pain. The disclosures of Foster et al. are incorporated in its entirety by reference herein.
In one embodiment, substantially similar methods to that of Foster et al. can be employed, but using the compositions described herein to treat pain. The pain to be treated can be an acute pain or chronic pain. An acute or chronic pain that is not associated with a muscle spasm can also be alleviated with a local, peripheral administration of the modified neurotoxin to an actual or a perceived pain location on the mammal.
In one embodiment, the BoNT polypeptide is administered subcutaneously at or near the location of pain, for example, at or near a cut. In some embodiments, the modified neurotoxin is administered intramuscularly at or near the location of pain, for example, at or near a bruise location on the mammal. In some embodiments, the BoNT polypeptide is injected directly into a joint of a mammal, for treating or alleviating pain caused by arthritic conditions. Also, frequent repeated injection or infusion of the modified neurotoxin to a peripheral pain location is within the scope of the present disclosure. Routes of administration for such methods are known in the art and easily adapted to the methods described herein by the skilled practitioner (e.g., see for example, Harrison's Principles of Internal Medicine (1998), edited by Anthony Fauci et al., 14.sup.th edition, published by McGraw Hill).
By way of non-limiting example, the treatment of a neuromuscular disorder can comprise a step of locally administering an effective amount of the molecule to a muscle or a group of muscles, the treatment of an autonomic disorder can comprise a step of locally administering an effective of the molecule to a gland or glands, and the treatment of pain can comprise a step of administering an effective amount of the molecule the site of the pain. In addition, the treatment of pain can comprise a step of administering an effective amount of a modified neurotoxin to the spinal cord.
“A therapeutically effective amount” as used herein refers to the amount of each therapeutic agent of the present disclosure required to confer therapeutic effect on the subject, either alone or in combination with one or more other therapeutic agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual subject parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, therapeutic agents that are compatible with the human immune system, such as polypeptides comprising regions from humanized antibodies or fully human antibodies, may be used to prolong half-life of the polypeptide and to prevent the polypeptide being attacked by the host's immune system.
Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a disease. Alternatively, sustained continuous release formulations of a polypeptide may be appropriate. Various formulations and devices for achieving sustained release are known in the art. In some embodiments, dosage is daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, dosing frequency is once every 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 longer. The progress of this therapy is easily monitored by conventional techniques and assays.
The dosing regimen (including the polypeptide used) can vary over time. In some embodiments, for an adult subject of normal weight, doses ranging from about 0.01 to 1000 mg/kg may be administered. In some embodiments, the dose is between 1 to 200 mg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular subject and that subject's medical history, as well as the properties of the polypeptide (such as the half-life of the polypeptide, and other considerations well known in the art).
For the purpose of the present disclosure, the appropriate dosage of a therapeutic agent as described herein will depend on the specific agent (or compositions thereof) employed, the formulation and route of administration, the type and severity of the disease, whether the polypeptide is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the antagonist, and the discretion of the attending physician. Typically the clinician will administer a polypeptide until a dosage is reached that achieves the desired result.
Administration of one or more polypeptides can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a polypeptide may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a disease. As used herein, the term “treating” refers to the application or administration of a polypeptide or composition including the polypeptide to a subject in need thereof.
“A subject in need thereof”, refers to an individual who has a disease, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease. In some embodiments, the subject has CDI. In some embodiments, the subject has cancer. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is human. Alleviating a disease includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results.
As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset.
As used herein “onset” or “occurrence” of a disease includes initial onset and/or recurrence. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the isolated polypeptide or pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
As used herein, a “subject” refers to a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human.
The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female. A subject can be a fully developed subject (e.g., an adult) or a subject undergoing the developmental process (e.g., a child, infant or fetus). Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously 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 domesticated animals and/or pets.
The following examples are intended to be illustrative of certain embodiments and are non-limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLES
Botulinum neurotoxins (BoNTs) are among the most dangerous potential bioterrorism agents and are also used clinically to treat a growing list of medical conditions. There are seven serotypes of BoNTs (BoNT/A-G) known to date, and no new types have been recognized for the past 45 years. Ggenomic database searching of Clostridium botulinum strains revealed a novel BoNT type, named BoNT/X. This toxin showed the lowest sequence identity with other BoNTs and it is not recognized by antisera raised against known BoNT types. It cleaves vesicle associated membrane protein (VAMP) in neurons, which is also the target of BoNT/B/D/F/G, but BoNT/X cleaves at a site (between Arg66-Ala67 on VAMP2) unique to this toxin. To validate the activity of BoNT/X, a limited amount of full-length BoNT/X were assembled by covalently linking two non-toxic fragments of BoNT/X using a transpeptidase (sortase). Assembled BoNT/X entered cultured neurons and cleaved VAMP2, and caused flaccid paralysis in mice measured by Digit Abduction Score assay. Together, these data established BoNT/X as a novel BoNT type. Its discovery poses an urgent challenge for developing effective countermeasures and also presents a novel tool for potential therapeutic applications.
Searching Genomic Databases Revealed a Novel BoNT GeneIn an attempt to survey the evolutionary landscape of BoNTs, iterative Hidden Markov model searches of the PubMed sequence database were performed, utilizing sequences of the seven BoNTs as probes. The search successfully identified major BoNT serotypes, subtypes, and mosaic toxins, as well as related tetanus neurotoxin (TeNT) (
Phylogenetic analysis revealed that BoNT/X is clear distinct from all other BoNTs and TeNT (
Despite the low sequence identity, the overall domain arrangement and a few key features of BoNTs appear to be conserved in BoNT/X (
As expected, BoNT/X gene is preceded with a putative NTNHA gene (
Whether BoNT/X is a functional toxin was next examined. First, the LC of BoNT/X (X-LC) was investigated. The border of the LC (residues 1-439) was determined by sequence alignment with other BoNTs. The cDNA encoding the LC was synthesized and the LC was produced as a His6-tagged recombinant protein in E. coli. X-LC was incubated with rat brain detergent extracts (BDE) and immunoblot analysis was used to examine whether the three dominant SNARE proteins in the brain, SNAP-25, VAMP2, and syntaxin 1, were cleaved. LCs of BoNT/A (A-LC) and BoNT/B (B-LC) were assayed in parallel as controls. Cleavage of SNAP-25 by BoNT/A generates a smaller fragment that can still be recognized on immunoblot, while cleavage of VAMP2 by BoNT/B abolishes the immunoblot signal of VAMP2 (
To identify the cleavage site on VAMP2, the VAMP2 (1-96) protein was analyzed with or without pre-incubation with X-LC, by liquid chromatography-tandem mass spectrometry (LC-MS/MS,
R66-A67 is a novel cleavage site distinct from established target sites for all other BoNTs (
Unexpectedly, Ykt6 was cleaved by X-LC (
A remarkable 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 trans-Golgi network (TGN) and endosomes, as well as homotypic fusion of endosomes59,60. Ykt6 is an atypical SNARE without a transmembrane domain67-70. It is anchored to membranes via lipidation, which allows dynamic regulation of its membrane association. Ykt6 is an essential protein in yeast, implicated in multiple membrane fusion events including ER-Golgi, intra-Golgi, endosome-Golgi-vacuolar, and autophagosome formation. Its function in mammalian cells remains to be established. BoNTs are traditionally known to be limited to target SNAREs that mediate vesicle exocytosis onto plasma membranes. BoNT/X is the first BoNT that is capable of cleaving SNAREs mediating various intracellular membrane trafficking events.
Interestingly, both VAMP4 and Ykt6 are enriched in neurons. Recent studies suggested that VAMP4 may also contribute to asynchronous synaptic vesicle exocytosis, enlargeosome exocytosis, and activity-dependent bulk endocytosis (ADBE) in neurons 61-63. The role of Ykt6 in neurons remains to be established, but it has been shown to suppress the toxicity of α-synuclein in Parkinson's disease models71-72. The other substrate of BoNT/X, VAMP5, is mainly expressed in muscle cells and its function remains to be established64. BoNT/X will be a powerful tool for investigating VAMP4, Ykt6, and VAMP5 functions and related membrane trafficking events. In addition, VAMP4 has been implicated in granule release in immune cells 65, thus BoNT/X might have a unique potential among all BoNTs to modulate inflammatory secretion in immune cells.
Proteolytic Activation of BoNT/XBoNTs are initially produced as a single polypeptide. The linker region between LC and HN needs to be cleaved by either bacterial or host proteases in a process known as “activation”, which is essential for the activity of BoNTs. LC and HN of BoNTs remain connected via an inter-chain disulfide bond prior to translocation of LC into the cytosol of cells, where the disulfide bond is reduced in order to release the LC into the cytosol. Sequence alignment revealed that BoNT/X contains the longest linker region between two conserved cysteines compared to all other BoNTs (C423-C467,
To examine whether the linker region between the LC and HN of BoNT/X is susceptible to proteolytic cleavage, a recombinant X-LC-HN fragment (residues 1-891) was produced in E. coli and subjected to limited proteolysis by endoproteinase Lys-C, which cuts at the C-terminal side of lysine residues. To identify the susceptible cleavage site under limited proteolysis conditions, X-LC-HN was analyzed using Tandem Mass Tag (TMT) labeling and tandem mass spectrometry approach. TMT labels free N-terminus (and lysines). Limited proteolysis by Lys-C produces additional free N-termini, which would not exist in intact X-LC-HN sample (see below for details). Briefly, intact X-LC-HN samples were labeled with the light TMT and equal amount of X-LC-HN samples were exposed to Lys-C and then labeled with the heavy TMT. Both samples were then digested with chymotrypsin, combined together, and subjected to quantitative mass spectrometry analysis. A list of identified peptides was shown in Table 2, below. The light TMT: heavy TMT ratios were usually within 2-fold of each other for each peptide, with the exception for 5 peptides starting with N439, which showed no signal for the light TMT labeling, indicating that this is a new N-terminal generated by Lys-C cutting (
Whether this proteolytic activation is important for the function of BoNT/X was examined next. It has been previously shown that incubation of high concentrations of LC-HN of BoNTs with cultured neurons resulted in entry of LC-HN into neurons, likely through non-specific uptake into neurons46,47. Using this approach, the potency of intact versus activated X-LC-HN on cultured rat cortical neurons was compared. Neurons were exposed to X-LC-HN in media for 12 hours. Cell lysates were harvested and immunoblot analysis was carried out to examine cleavage of SNARE proteins. As shown in
The linker region of BoNT/X contains an additional cysteine (C461), which is unique to BoNT/X. To determine which cysteine forms the disulfide bond connecting the LC and HC, three X-LC-HN mutants were generated, with each of the three cysteine residues mutated (C423S, C461S, and C467S). These three cysteine mutants, as well as wild type (WT) X-LC-HN were subjected to limited proteolysis and then analyzed via SDS-PAGE and Coomassie Blue staining, with or without reducing agent DTT (
The majority of activated WT X-LC-HN also separated to two ˜50 kDa bands on SDS-PAGE gel without DTT (
Finally, the activity of the three X-LC-HN cysteine mutants on cultured neurons was examined. As shown in
To evaluate whether BoNT/X is a functional toxin, it was necessary to generate and test full-length BoNT/X. However, BoNTs are one of the most dangerous potential bioterrorism agents. Therefore, the necessary precaution was taken, and the full-length active toxin gene was not generated. Instead, an approach to generate limited amounts of full-length BoNTs in test tubes under controlled conditions by the enzymatic ligation of two non-toxic fragments of BoNTs was developed. This method utilizes a transpeptidase known as sortase, which recognizes specific peptide motifs and covalently link two peptides together by forming a native peptide bond (
An engineered sortase A, known as SrtA*, from Staphylococcus aureus was generated51. SrtA* recognizes the peptide motif LPXTG (SEQ ID NO: 57), cleaves between T-G, and concurrently forms a new peptide bond between the protein containing LPXTG (SEQ ID NO: 57) with other proteins/peptides containing one or more N-terminal glycine (
It was observed that X-HC showed a strong tendency for aggregation in solution for unknown reasons once it is cut from GST tag, which might be the reason why the ligation efficiency is low for BoNT/X (
To analyze the activity of full-length BoNT/X, cultured rat cortical neurons as a model system were used. Neurons were exposed to the sortase ligation mixture and various control mixtures in media. Cell lysates were harvested 12 hours later and immunoblot analysis was carried out to examine cleavage of SNARE proteins. As shown in
Mixing X-HC with X-LC-HN increased the amounts 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-He formed inter-molecular disulfide bond with X-LC-HN. The presence of DTT also increased the amount of ligated full-length BoNT/X, suggesting that a portion of BoNT/X aggregated via inter-molecular disulfide bond (
Whether BoNT/X is active in vivo was examined using a well-established non-lethal assay in mice, known as Digit Abduction Score (DAS). This assay measures local muscle paralysis following injection of BoNTs into mouse hind limb muscles52,53. BoNTs cause flaccid paralysis of limb muscles, which can be detected by the failure to spread the toes during the startle response. An activated sortase reaction mixture (
To further confirm that BoNT/X is a serologically unique BoNT, dot blot assays were carried out using antisera raised against known BoNTs, including all seven serotypes as well as one mosaic toxin (BoNT/DC). Four horse antisera were utilized (trivalent anti-BoNT/A, B, and E, anti-BoNT/C, anti-BoNT/DC, and anti-BoNT/F), as well as two goat antisera (anti-BoNT/G and anti-BoNT/D). These antisera were all capable of neutralizing their corresponding target BoNTs and prevented cleavage of SNARE proteins in neurons (
Finally, whether full-length BoNT/X can be produced as a soluble protein was examined. To ensure the biosafety requirement, mutations in the LC of BoNT/X were introduced that inactivate its toxicity. Mutations at two residues R362A/Y365F in BoNT/A have been shown to inactivate the protease activity of the LC in vitro and abolishes the toxicity of full-length BoNT/A in mice in vivo54-56. These two residues are conserved in all BoNTs including BoNT/X. Therefore, the corresponding mutations were introduced at these two sites (R360A/Y363F in BoNT/X). As shown in
A substantial portion of BoNT/XRY formed aggregates at the top of the SDS-PAGE gel (
An inactive form of BoNT/X might be utilized as a vehicle to deliver therapeutics into neurons. Inactivation can be achieved by mutations at any one of the following residues or their combinations: R360, Y363, H227, E228, or H231, with the later three residues forming the conserved protease motif.
Purification of Full-Length Inactive BoNT/X at Industrial-ScaleWhether full-length BoNT/X can be purified to a high degree of purity and with a good yield, which will be important for industrial production of BoNT/X (or its derivative) as a therapeutic toxin, was investigated. Several parameters of cell growth and expression were tested, such as temperature, time of induction and IPTG concentrations. The optimal parameters chosen for protein expression were culture of the cells at 37° C. until they reached exponential growth, at which stage the temperature was reduced to 18° C. and expression induced by addition of 1 mM IPTG to the media. Cells were then cultured for 16 to 18 hours before harvesting. Presence of BoNT/X was verified by SDS-PAGE and showed a high level of over-expression in the soluble fraction (
Several small-scale purification trials were carried out to optimize the production process. Mechanical cell lysis using an Emulsiflex-C3 (Avestin, Mannheim, Germany) was the preferred method for intracellular protein extraction, and appeared more efficient that sonication. Various buffer conditions also had to be assessed for optimal recovery of BoNT/X. A reducing agent was included throughout the purification process and greatly decreased the propensity to unwanted aggregation. Additionally, glycerol was used as an additive during the early stage of the purification process and improved protein stability.
The BoNT/X construct was expressed with a HIS6-tag that could be used for affinity chromatography as a first purification step. For small-scale trials, a 5 ml HIsTrapFF column (GE Healthcare, Danderyd, Sweden) was used. In order to achieve the highest purity from the initial chromatography, various concentrations of imidazole were tested. BoNT/X eluted from a concentration of 100 mM imidazole; however, a major contaminant readily co-purified with the toxin. This contaminant appeared to non-specifically interact with BoNT/X and was identified by mass spectrometry as an E. coli host protein (bifunctional polymyxin resistance protein AmA). The presence of this contaminant was dramatically reduced with the introduction of a high salt concentration (500 mM NaCl) and by carrying out an additional washing step at 100 mM imidazole during purification. This allowed for elution of a purer BoNT/X fraction at 250 mM imidazole. This later fraction could then be polished by size exclusion chromatography.
Once in place, this protocol was scaled up by expressing up to 12 L of media with the conditions described above. Additionally, a larger affinity chromatography matrix was prepared consisting of 15 ml of Protino® Ni-NTA agarose (Macherey-Nagel, Duren, Germany) to increase the yield of BoNT/X recovery. The final purification step was performed by size exclusion chromatography using a Superdex200-16/60 column (GE Healthcare, Danderyd, Sweden). Using this method, between 85 and 90% purity was obtained (
Note that the purification was done in the presence of reducing agent, which would reduce the disulfide bond between the LC and the HC, so purified toxin would not be active. However, a designed BoNT/X derivative containing mutations at the cysteine sties (one mutation at C461 or C467, combined with mutating C1240) would be able to be purified without reducing agents. Note that an inactive form of BoNT/X (and its cysteine mutation derivative) might be utilized as a vehicle to deliver therapeutics into neurons. Inactivation can be achieved by mutations at any one of the following residues or their combinations: R360, Y363, H227, E228, or H231 (the later three residues form the conserved protease motif).
Identification of Gangliosides as Receptors for BoNT/XGangliosides are well-established lipid co-receptors for all BoNTs and a ganglioside-binding motif is well-conserved in BoNT/X (
The eighth serotype of BoNTs over 45 years after the identification of the last major BoNT serotype has been identified. BoNT/X has the lowest protein sequence identity to any other BoNTs and TeNT among this family of toxins, and this low level of identity is evenly distributed along the toxin sequence. 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 revealed by searching genomic sequences of clostridium botulinum strains and it represents the first major toxin type identified by genomic sequencing and bioinformatics approach. The strain 111 that contains BoNT/X gene was initially identified from an infant botulism patient in 1990s. Previous characterizations using classic neutralization assay have established BoNT/B2 as the major toxin of this strain. It is likely that BoNT/X is a silent toxin gene, or it was not expressed at detectable toxicity levels under the culture conditions in the lab. Therefore, it can only be identified by sequencing strain 111. This illustrates the importance of genomic sequencing and bioinformatics approaches for understanding microbial virulent factors.
Silent BoNT genes have been frequently found previously in various Clostridium botulinum strains. It is not clear why these bacteria keep a silent toxin gene. It could be an evolutionarily degenerated gene. This is clearly the case when silent toxin genes contain premature stop code mutations. However, there are also cases that the silent gene encodes a full-length BoNT. Whether these silent full-length BoNTs might be expressed and exhibit toxicity under certain environmental conditions remains an intriguing question.
The general three-domain structures and functions of BoNTs are well conserved in BoNT/X, but it also has a few unique characteristics: (1) it shares VAMP as its target in neurons with BoNT/B, D, F, and G, but it cuts VAMP at a novel site (R66-A67 in VAMP2) that is unique to this toxin. This further expands the repertoire of toxins that can be used to ablate VAMP at different sites. (2) The inter-chain disulfide bond connecting LC and HN is conserved in BoNT/X, but it also contains a unique additional cysteine in the linker region, which may lead to disulfide bond shuffling. The extra cysteine on HN is not essential for the activity of LC-HN (
His6-tagged X-LC-HN fragment are stable in buffers as recombinant proteins. It showed a higher level of activity on neurons than both A-LC-HN and B-LC-HN (
X-HC is functional as its presence enhanced cleavage of VAMP2 in neurons than LC-HN alone (
Gangliosides have long been established as neuronal receptors for all BoNT subtypes. It is demonstrated that BoNT/X can bind to all four of the most abundant gangliosides: GD1a, GD1b, GT1b, and GM1. Additionally it does so with remarkable difference in affinity and specificity when compared to BoNT/A. This is an intriguing property, as other BoNTs appear to have various degrees of preferences toward a subgroup of gangliosides. For instance, BoNT/A, E, F, and G prefer GD1a and GT1b. BoNT/X might potentially recognize a broader range of neuron types compared to other BoNTs.
It is possible that BoNT/X has a low toxicity in vivo, which might explain why BoNT/X activity was not detected in the original study on strain 111. If this is the case, the reduced toxicity is likely due to its HC domain, as X-LC-HN appears to be more active than both A-LC-HN and B-LC-HN. The formation of inter-molecular disulfide bond might also reduce the effective toxin concentration. It will be necessary to produce full-length native BoNT/X in order to determine its potency in vivo, but it will be important to generate neutralizing antisera using non-toxic fragments of BoNT/X prior to producing full-length toxin.
Introducing full-length active toxin gene into any expression systems/organisms is always a significant biosafety concern and it has become a formidable hurdle for structure-function studies of biological toxins. This is particularly an important consideration for BoNTs as they are one of the six category A potential bioterrorism agents 4. Here a method to assemble limited amount of full-length toxin biochemically from two complementary and non-toxic fragments was developed. Each fragment is expressed and purified individually, and then ligated together by sortase in test tubes. Other protein ligation methods such as split intein systems, which fuse two protein fragments through protein trans-splicing, can also be utilized57. By controlling the amount of precursor fragments in the reaction, the amount of ligated full-length toxin can be strictly controlled. This “semi-synthesis” approach can be used to produce multi-domain biological toxins and other toxic proteins under controlled conditions. It also provides a versatile platform for generating fusion and chimeric toxins, such as swapping the HC of two BoNTs, replacing HC of BoNTs with other targeting proteins, or attaching additional cargo to toxins. As there is no full-length toxin cDNA ever generated and no expression of toxins in bacteria or any other living organisms, this approach significantly mitigates the biosafety concerns associated with producing wild type and mutant toxins and will greatly facilitate structure-function studies of biological toxins and toxic proteins.
Materials and MethodsMaterials: Mouse monoclonal antibodies for syntaxin 1 (HPC-1), SNAP-25 (C171.2), and VAMP2 (C169.1) were generously provided by E. Chapman (Madison, Wis.) and are available from Synaptic Systems (Goettingen, Germany). Mouse monoclonal antibody for actin was purchased from Sigma (AC-15). Equine polyclonal antisera against BoNT/A/B/E, BoNT/C, BoNT/DC, BoNT/F, and goat polyclonal antisera against BoNT/G were obtained from the FDA. Goat polyclonal antibody against BoNT/D 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, Wis.). BoNT/D was generously provided by E. Johnson (Madison, Wis.).
cDNA and constructs: The cDNAs encoding X-LC (residues 1-439) and X-HC (residues 893-1306) was synthesized. The cDNA encoding X-HN was generated in-house using Gibson assembly method. The cDNAs encoding A-LC (residues 1-425, M30196) and B-LC (residues 1-439, AB232927) were synthesized by GenScript (New Brunswick, N.J.). These LCs were cloned into pET28 vectors for expression as His6-tagged proteins. X-HC was cloned into pGEX4T to express as a GST-tagged protein. X-LC-HN, A-LC-HN, and B-LC-HN were subcloned into pET28 vector, with a peptide sequence LPETGG (SEQ ID NO: 58) fused to their C-termini, and were purified as His6-tagged proteins. Full-length inactive form of BoNT/X was assembled in-house from mutated X-LC (R360A/Y363F), X-HN, and X-HC. It was cloned into pET28 vector with a His6-tagg fused to the C-terminus of BoNT/X. The cDNA encoding rat VAMP2 was generously provided by E. Chapman (Madison, Wis.). VAMP2 (1-96) was cloned into pET28 vector and expressed as a His6-tagged protein. VAMP2 (33-86) was cloned into a pGEX4T vector and expressed as a GST-tagged protein. The cDNA encoding mouse VAMP1, VAMP3, rat VAMP7, and VAMP8 was generously provided by C. Hu (Louisville, Ky.). They were cloned into a modified pcDNA3.1 vectors, with a HA tag fused to their C-termini. The construct encoding His6-tagged sortase (SrtA*) was generously provided by B. Pentelute (Boston, Mass.) and has been described previously51.
Bioinformatics: The Uniprot database was searched with jackhmmer at the HMMER web server using a BoNT type A sequence (Uniprot accession number A5HZZ9) until convergence. Returned sequences were aligned with Clustal Omega and a NeighborNet phylogenetic network estimated with SplitsTree4.
Protein purification: E. coli BL21 (DE3) was utilized for protein expression. Induction of expression was carried out 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 20000 g for 30 min at 4° C. Protein purification was carried out using AKTA Prime FPLC system (GE) and purified proteins were further desalted with PD-10 column (GE, 17-0851-01). Specifically, full-length inactive BoNT/X (BoNT/XRY) was cloned into a pET22b vector. The corresponding plasmid was transformed into E. coli BL21 (DE3) competent cells. Resulting colonies were used to inoculate 100 ml overnight cultures of TB medium containing 100 μg/ml Carbenicillin in 250 ml shake-flask and grown at 37° C. Cultures for expression were first grown using a LEX Bioreactor (Epiphyte3, Ontario, Canada) at 37° C. in 1.5 L of TB media until OD600 reached 0.8. The temperature was then reduced to 18° C. for induction of expression with 1 mM IPTG, and grown for 16-17 hours. Cells were harvested and re-suspended on ice in 50 mM HEPES pH 7.2, 500 mM NaCl, 25 mM imidazole, 5% glycerol, 2 mM TCEP to allow for cell lysis with an Emulsiflex-C3 (Avestin, Mannheim, German) at 20,000 psi. Lysate was ultra-centrifuged at 200,000 g for 45 minutes at 4° C. Supernatnt was loaded onto a 15 ml Protino@ Ni-NTA agarose (Macherey-Nagen, Diren, Germany) column that was then washed with 50 mM HEPES pH 7.2, 500 mM NaCl, 100 mM imidazole, 5% glycerol, 1 mM TCEP. Elution was carried out 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. Dialysate was concentrated using a Vivaspin concentrator (100 kDa cut-off, GE Healthcare, Danderyd, Sweden) before being loaded on a Superdex200-16/60 column (GE Healthcare, Danderyd, Sweden) pre-equiliberated in the same buffer as was used for dialysis. The elution peak corresponding to BoNT/X was collected and concentrated so that the final sample was at 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 in methanol to reach a final concentration of 2.5 μg/ml; 100 μL was applied to each well of a 96-well PVC assay plate (catalog no. 2595, Corning; Corning, N.Y.). After solvent evaporation at 21° C., the wells were washed with 200 μL PBS/0.1% (w/v) BSA. Nonspecific binding sites were blocked by incubation for 2.5 h at 4° C. in 200 μL of 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. containing samples (triplicate) at concentrations ranging from 6 μM to 0.05 μM (in serial 2-fold dilution). Following incubation, wells were washed three times with PBS/0.1% (w/v) BSA and incubated with an HRP-conjugated anti-6×His monoclonal antibody (1:2000, ThermoFisher) for 1 h at 4° C. After three washing steps with PBS/0.1% (w/v) BSA, bound samples were detected using Ultra-TMB (100 μL/well, ThermoFisher) as the substrate. The reaction was stopped after 15 minutes by addition of 100 μL 0.2M H2SO4, and the absorbance at 450 nm was measured using an Infinite M200PRO plate reader (Tecan, Mannedorf, Switzerland). Data were analyzed with Prism7 (GraphPad Software).
Cleavage of SNARE proteins in rat brain detergent extracts (BDE): Rat BDE were prepared from fresh dissected adult rat brains as previously described 58. Briefly, a rat brain was homogenized in 15 ml 320 mM sucrose buffer, followed by a centrifugation at 5000 rpm for 2 min at 4° C. Supernatants were collected and centrifuged at 11,000 rpm for 12 min. The pellet was collected and solubilized for 30 min in 15 ml Tris-buffered saline (TBS: 20 mM Tris, 150 mM NaCl) plus 2% of Triton X-100 and a cocktail of protease inhibitors (Roche, CA). Samples were subsequently centrifuged at 17,000 rpm for 20 min to remove the insoluble materials. The final BDE concentration is ˜2 mg/ml proteins. BDE (60 μl) were incubated with X-LC (0.5 μM), A-LC (1 μM), or B-LC (1 μM), respectively, for 1 hour at 37° C., and were then analyzed by immunoblot using the enhanced chemiluminescence (ECL) method (Pierce). As controls, LCs were pre-incubated with 20 mM EDTA for 20 minutes at room temperature (RT) to de-active their activity prior to adding into 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 gels and Coomassie Blue staining, or subjected to mass spectrometry analysis.
Cleavage of VAMPs in cell lysates: Full-length HA-tagged VAMP1, 3, 7, and 8 were transfected into HEK293 cells using PolyJet transfection reagents (SignaGen, MD) following the manufacturer's instruction. Cell lysates were harvested 48 hours later in RIPA 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 hour at 37° C. Samples were then analyzed by immunoblot.
Whole Protein Analysis by LC-MS/IIMS: Samples were analyzed at Taplin Biological Mass Spectrometry Core Facility at Harvard Medical School. Briefly, whole protein samples were loaded onto a 100 μm internal diameter C18 reverse phase HPLC column packed with 3 cm of beads off-line using a pressure cell. The column was re-attached to an Accela 600 Pump (Thermo Fisher Scientific, Waltham, Mass.). A rapid gradient of increasing acetonitrile was used to elute the protein/peptide from the HPLC column. As peptides eluted, they were subjected to electrospray ionization and then entered into an LTQ Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, Mass.) to acquire a high resolution FTMS scan at 60000 resolution, a second scan at low resolution in the ion trap, and a final scan to perform data dependent MS/MS. The charge state envelopes were de-convoluted manually to obtain mono-isotopic masses when possible or average masses for the proteins. Peptide and protein identity were determined by matching protein databases with the acquired fragmentation pattern by the software program, Sequest (Thermo Fisher Scientific). All databases include a reversed version of all the sequences and the data was filtered to between a one and two percent peptide false discovery rate.
Identification of the protease cleavage site between LC and HN: His6-tagged recombinant X-LC-HN fragment (residues 1-891) was purified in E. coli and subjected to limited proteolysis by endoproteinase Lys-C(Sigma P2289, 100:1 (toxin:Lys-C) molar ratio, 25 minutes at room temperature). The cleavage site was determined by Tandem Mass Tag (TMT) labeling and tandem mass spectrometry approach. Briefly, intact X-LC-HN samples were labeled with the light TMT and equal amount of X-LC-HN samples were exposed to Lys-C and then labeled with the heavy TMT. Both samples were then digested with chymotrypsin, combined together, and subjected to quantitative mass spectrometry analysis.
Cysteine alkylation by NEM: Lys-C activated X-LC-HN fragment was diluted into sodium phosphate buffer (10 mM, pH 6.5) at the final concentration of 0.3 mg/ml, with or without NEM at indicated concentrations (20, 10, and 5 mM) and incubated for 10 minutes at RT. NEM was freshly prepared in sodium phosphate buffer. Samples were mixed with 3× neutral loading dyes (200 mM Tris pH 6.8, 30% glycerol, 6% Lithium Dodecyl sulfate, 10 mM NEM and 0.06% BPB). For samples without NEM, the same 3×SDS loading dye without NEM was used. Samples were further incubated with the loading dye at RT for 10 minutes, heated for 10 min at 55° C., and then analyzed by SDS-PAGE and Coomassie Blue staining.
Neuron culture and immunoblot analysis: Primary rat cortical neurons were prepared from E18-19 embryos using a papain dissociation kit (Worthington Biochemical, NJ), as we described previously 58. Experiments were carried out on DIV 14-16. Neurons were exposed to BoNT/X fragments or sortase ligation mixture in media for 12 hrs. 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). 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: BoNTs (0.2 μg in 1 μl) were spotted onto nitrocellulose membranes and dried (10 minutes at room temperature). The membranes were blocked with 5% milk in TBST (TBS plus 0.05% Tween20) for 30 min and then incubated with indicated antisera (1:500 dilution) for 30 min. The membranes were then washed three times with TBST and incubated with HRP (horseradish peroxidase) conjugated secondary antibodies for 30 min, washed three more times with TBST, and analyzed with the ECL method (Pierce). We note that the BoNT/X sample was composed of purified X-LC-HN and X-HC at 1:1 ratio.
Sortase-mediated ligation: GST-X-HC was cleaved overnight at 4° C. by thrombin before adding into the mixture of proteins. Ligation reaction was set up in 50 μl TBS buffer with addition of X-LC-HN (8 μM), thrombin-cleaved GST-X-HC (25 μM), Ca2+ (10 mM), and sortase (10 μM), for 40 min at RT. In
DAS assay: Sortase ligation mixture was first activated with limited proteolysis using trypsin (60:1 molar ratio (the total amount of the proteins:trypsin), 30 min at RT). We chose trypsin instead of Lys-C here as we can stop the proteolysis by adding trypsin inhibitor (Soybean trypsin inhibitor, 1:10 ratio (trypsin:trypsin inhibitor). Mice (CD-1 strain, 21-25 g, n=6) were anesthetized with isoflurane (3-4%) and were injected with sortase ligation mixture using a 30-gauge needle attached to the sterile Hamilton Syringes, into the gastrocnemius muscles of the right hind limb. BoNTs result in paralysis of the hind paw in the startle response. Muscle paralysis was observed within 12 hours after the injection as previously described52,53.
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All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
EQUIVALENTS AND SCOPEWhile several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted 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 “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. 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 refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
Claims
1.-175. (canceled)
176. A chimeric Clostridial Botulinum neurotoxin (BoNT) polypeptide comprising:
- (a) a protease domain;
- (b) a linker region;
- (c) a translocation domain; and
- (d) a receptor binding domain,
- wherein (a) and (c) are from BoNT serotype X, and wherein (d) is not from BoNT serotype X.
177. The chimeric BoNT polypeptide of claim 176, wherein the receptor binding domain is modified.
178. The chimeric BoNT polypeptide of claim 177, wherein the receptor binding domain comprises a substitution mutation corresponding to C1240S or C1240A in SEQ ID NO: 1.
179. The chimeric BoNT polypeptide of claim 176, wherein the receptor binding domain is from serotype selected from the group consisting of A, B, C, D, E, F, and G.
180. The chimeric BoNT polypeptide of claim 176 comprising an amino acid sequence that has at least 85% identity to any one of SEQ ID NOs: 22-24, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
181. The chimeric BoNT polypeptide of claim 180, comprising an amino acid sequence of any one of SEQ ID NOs: 22-24.
182. The chimeric BoNT polypeptide of claim 176, wherein the linker region comprises an artificial linker.
183. The chimeric BoNT polypeptide of claim 182, wherein the artificial linker comprises a cleavage site of a protease.
184. The chimeric BoNT polypeptide of claim 183, wherein the protease is selected from the group consisting of thrombin, TEV, PreScission (3C protease), Factor Xa, Enterokinase, and SUMO protease.
185. The chimeric BoNT polypeptide of claim 184, wherein the linker comprises an amino acid sequence of any of SEQ ID NOs: 50-56.
186. The chimeric BoNT polypeptide of claim 176, comprising one or more substitution mutation(s) in a position corresponding to R360, Y363, H227, E228, or H231 in SEQ ID NO: 1.
187. The chimeric BoNT polypeptide of claim 186, wherein the one or more substitution mutation(s) corresponds to R360A/Y363F, H227Y, E228Q, or H231Y in SEQ ID NO: 1.
188. A chimeric molecule comprising a first portion linked to a second portion, wherein the first portion is a chimeric BoNT polypeptide of claim 186.
189. The chimeric molecule of claim 188, wherein the first portion and the second portion are linked covalently.
190. The chimeric molecule of claim 189, wherein the first portion and the second portion are linked non-covalently.
191. The chimeric molecule of claim 189, wherein the second portion is selected from the group consisting of a small molecule, a nucleic acid, a short polypeptide and a protein.
192. The chimeric molecule of claim 191, wherein the second portion is a bioactive molecule.
193. The chimeric molecule of claim 191, wherein the second portion is a non-polypeptide drug.
194. A method of treating a condition associated with overactive neurons or glands, the method comprising administering a therapeutically effective amount of the modified BoNT polypeptide of claim 176.
195. The method of claim 194, wherein the condition is selected from the group consisting of: spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain from muscle spasms, headache pain, dermatological or aesthetic/cosmetic conditions, obesity/reduced appetite.
Type: Application
Filed: Dec 20, 2021
Publication Date: Jun 9, 2022
Applicant: Children's Medical Center Corporation (Boston, MA)
Inventors: Min Dong (Weatogue, CT), Sicai Zhang (Boston, MA), Paul Stenmark (Stockholm)
Application Number: 17/555,570
