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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
  • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
  • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
  • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site

Definitions

• the present invention relates to an engineered botulinum neurotoxin serotype E (BoNT/E), wherein multiple amino acid sequences have been substituted into the Heavy Chain Binding domain (He) forming a synaptic associated vesicle 2C (SV2C) receptor binding site in the BoNT/E H c .
• BoNT/E botulinum neurotoxin serotype E
• He Heavy Chain Binding domain
• SV2C synaptic associated vesicle 2C
• BoNTs Botulinum neurotoxins
• BoNTs are a family of proteins produced mainly by Clostridium botulinum. BoNTs are expressed as a single ⁇ 150 kDa polypeptide that is subsequently cleaved into an active di-chain molecule consisting of a ⁇ 50 kDa light-chain (LC) and a ⁇ 100 kDa heavy- chain (HC) (Swaminathan, 2011). The LC and HC remain bound by a single disulphide-bond.
• LC light-chain
• HC heavy-chain
• the LC comprises a single zinc-dependent protease domain and the HC comprises two distinct domains, the translocation domain (H N ) and receptor-binding domain (He).
• BoNTs target and block synaptic vesicle exocytosis at neuronal terminals which results in paralysis of the associated muscle (Dong, Masuyer & Stenmark, 2019). This is achieved through a three-step intoxication process; first the He targets and binds to receptors on the neuronal cell surface. The toxin is then able to enter the cell by endocytosis where it remains within the early endosome. Here the H N enables transport of the LC across the endosomal membrane and into the cytosol.
• the LC cleaves a SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) protein resulting in the inhibition of SNARE-mediated exocytosis, halting the release of acetylcholine into the neuromuscular junction (Dong, Masuyer & Stenmark, 2019).
• SNARE soluble N-ethylmaleimide-sensitive-factor attachment protein receptor
• BoNT serotypes [004] Several different BoNT serotypes have been identified on the basis of neutralization using specific antisera (Schiavo, Matteoli & Montecucco, 2000). Serotypes A through G (BoNT/A - /G) are well established, while more recently several new BoNT serotypes have been identified including BoNT/X, BoNT/En, and BoNT/Wo (Tanizawa et al., 2014; Zornetta et al., 2016; Zhang et al., 2017, 2018).
• BoNT serotypes bind both a protein receptor and a polysialoganglioside (Rummel, 2017).
• BoNTs bind one of two possible protein receptors: synaptotagmin or synaptic vesicle glycoprotein 2 (SV2).
• SV2 is the receptor for BoNT/A, /E, /D, and /F (Dong et al., 2006; Mahrhold et al., 2006; Dong et al., 2008; Rummel et al., 2009; Peng et al., 2011).
• SV2 proteins are produced as one of three isoforms (SV2A, SV2B, SV2C), each of which are glycosylated and contain 12 transmembrane regions along with two globular loops, one luminal and the other cytoplasmic (Bartholome et al., 2017).
• the expression levels of each SV2 isoform differs between different neuronal cell types and even sub-populations within a particular cell type.
• SV2C is expressed at greater levels than either SV2A or SV2B in motor neurons (Pellett, Tepp & Johnson, 2019).
• BoNT/A subtypes can utilise all three SV2 isoforms to enter neuronal cells, the preferred isoform with the greatest affinity is SV2C.
• BoNT/E binds predominantly SV2A but also SV2B (Dong et al., 2008; Rummel et al., 2009).
• SV2A is found in relatively low levels within motor neurons however it is upregulated within motor neurons which are associated with slow-type muscle fibres (Chakkalakal et al., 2010).
• BoNT/E is capable of translocating its LC faster than that of BoNT/A (Wang et al., 2008).
• An increased translocation rate is one factor which determines the onset time.
• the mechanism of translocation is not yet understood but it is likely to involve a conformational change of the H N which forms a full or partial pore, allowing an unfolded LC through (Pirazzini et al., 2015).
• the crystal structure of BoNT/E revealed a domain organisation different from what was previously observed for BoNT/A.
• the different domain organisation of BoNT/E, when compared to BoNT/A might be responsible for the enhanced translocation rate observed (Lacy et al., 1998; Lacy & Stevens, 1999; Kumaran et al., 2009).
• BoNTs botulinum neurotoxins
• Many medical and cosmetic applications would benefit from a faster onset of action.
• the most widely used commercial BoNT has an onset time of approximately 2-3 days.
• An alternative toxin serotype, BoNT/E is currently being explored for its fast onset time of approximately 12 hours.
• BoNT/E targets only a subset of the available receptors found on neuronal cells (SV2A and SV2B) which does not include SV2C, compared with BoNT/A which can utilise all isoforms (SV2A, SV2B, SV2C).
• BoNT/E may have a faster onset rate than BoNT/A but it is unable to utilise SV2C which may potentially reduce its potency.
• the design of a toxin which combines the action of BoNT/E with the potency of BoNT/A would be highly useful for the therapeutic market.
• the chimeras formed by Wang et al. do not take into account the potential interaction between the He and the H N domains, which may be important for overall toxin stability and during toxin translocation.
• the ganglioside- binding site of the BoNT/E may also be modified.
• it would be desirable with a BoNT chimera that ensures that any potential interface between the Hcand H N domains are maintained and that the ganglioside-binding site from BoNT/E is maintained.
• BoNT/E-like toxin based on the native sequences of BoNT/A and BoNT/E, which BoNT/E-like toxin recognises SV2C and binds with greater affinity to SV2C than native BoNT/E.
• the BoNT/E chimera preferably having a maintained interface between the Hcand HN domains and a maintained ganglioside-binding site.
• the novel BoNT/E - BoNT/A chimeras being produced using an advanced three- dimensional structural design process.
• This object is thus attained by in a first aspect providing a modified/engineered botulinum neurotoxin serotype E (BoNT/E) Heavy Chain Binding domain (He) (SEQ ID NO: 3) comprising multiple amino acid substitutions in the BoNT/E He sequence (SEQ ID NO: 2) forming a synaptic associated vesicle 2C (SV2C) receptor binding site in the modified BoNT/E He, wherein the multiple amino acid substitutions in the modified BoNT/E He sequence comprise the following substitutions with the amino acid residue numbering of the non- modified BoNT/E full length amino acid sequence (SEQ ID NO: 7): substitution 2a or substitution 2b, wherein substitution 2a replaces amino acids at positions 925-933 and comprises an amino acid sequence: Lys Tyr Phe Asn Ser lie Ser Leu, and substitution 2b replaces amino acids at positions 925-932 and comprises an amino acid sequence: Lys Tyr Phe Asn Ser lie Ser; substitution 3, wherein substitution 3 replaces amino acids 956-957 and
• substitution 2a could be replaced with its shorter counterpart substitution 2b. It is believed, however, that the longer substitution 2a, results in a BoNT/E He chimera that binds better to the SV2C receptor than a BoNT/E He chimera with the trimmed substitution 2b.
• the modified BoNT/E He may further comprise the following substitutions with the amino acid residue numbering of the non-modified BoNT/E full length amino acid sequence: substitution 1, wherein substitution 1 replaces amino acid 891-896 and comprises an amino acid sequence: Phe Asn Leu Glu Ser Ser, and/or substitution 6, wherein substitution 6 replaces amino acid 1097 and comprises amino acid: Tyr.
• the modified BoNT/E He comprising also one or both of these substitutions may bind better to the SV2C receptor than a modified BoNT/E He without these additional substitutions.
• the BoNT/E H c may be selected from any subtype El, E2, E3, E4, E5, E6, E7, E8, E9, E10, Ell or E12.
• BoNT/E subtypes share between 88% and 99% amino acid sequence identity. It is possible that not yet identified BoNT/E subtypes will be revealed in the future. The identified substitutions discussed above grafted into such BoNT/E He would then also most likely result in BoNT/E He chimeras with binding affinity for the SV2C receptor.
• a polypeptide comprising the modified BoNT/E He described above coupled to any one or more of a protein, a polypeptide, an amino acid sequence, or a fluorescent probe directly or via a linker.
• Such polypeptide is preferably produced recombinantly as the BoNT/E He needs to be produced recombinantly.
• the nucleic acids encoding the modified BoNT/E He or polypeptide comprising the modified BoNT/E He may be DNA or RNA, double-stranded or single stranded.
• the protein to be included in the polypeptide may be any protein of interest to be transported to a neuronal cell, and/or internalized into a neuronal cell.
• the polypeptide may in addition to the He comprise a Heavy Chain Translocation domain (HN), a Light chain (LC) and a protease site positioned between the LC and H N in the polypeptide sequence, wherein the H N and the LC, respectively and independently originate from any of the BoNT serotypes A, B, C, D, DC, E, En, F, G, Wo or X or their subtypes.
• HN Heavy Chain Translocation domain
• LC Light chain
• protease site positioned between the LC and H N in the polypeptide sequence, wherein the H N and the LC, respectively and independently originate from any of the BoNT serotypes A, B, C, D, DC, E, En, F, G, Wo or X or their subtypes.
• a vector comprising a nucleic acid sequence encoding the modified BoNT/E He or the polypeptide described above.
• the vector may further comprise a nucleic acid sequence encoding any other protein or probe that is to be recombinantly produced together with the modified BoNT/E He, so as to obtain said protein or probe coupled to the modified BoNT/E He in one polypeptide.
• the vector is preferably an expression vector.
• the vector may comprise a promoter operably linked to the nucleic acid. A variety of promoters can be used for expression of the polypeptides described herein, and are known to the person skilled in the technical field.
• 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.
• the expression of the polypeptides described herein is regulated by a constitutive, an inducible or a tissue-specific promoter.
• the polypeptides may be produced in any cells, eukaryotic or prokaryotic, or in yeast.
• the polypeptides according to the invention may further be produced in a cell free system.
• the modified BoNT/E He and/or the polypeptide may be used in a therapeutic or cosmetic method.
• the modified BoNT/E He and/or the polypeptide targets SV2C and has a higher affinity than native/non-modified BoNT/E for SV2C.
• the therapeutic treatment or cosmetic treatment may be a treatment to dampen and/or inactivate muscles.
• the modified BoNT/E He or the polypeptide may preferably be used to prevent and/or treat wrinkles, brow furrows or unwanted lines, in order to reduce said wrinkles, furrows and lines.
• the therapeutic method may be a treatment and/or prevention of a disorder chosen from the group comprising neuromuscular disorders and spastic muscle disorders.
• the disorder may be chosen from the group comprising 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, sports injuries, and depression.
• a pharmaceutical or cosmetic composition comprising the modified BoNT/E He and/or the polypeptide.
• the modified BoNT/E He and/or peptide according to the above may be formulated in any suitable pharmaceutical or cosmetic composition.
• the pharmaceutical composition may further comprise pharmaceutically acceptable excipients, carriers or other additives.
• the cosmetic composition comprising the modified BoNT/E He and/or peptide may further comprise cosmetically acceptable excipients, carriers or other additives.
• compositions for administration by injection are solutions in sterile isotonic aqueous buffer.
• the composition can also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
• a method of treating a condition associated with unwanted neuronal activity comprising administering a therapeutically effective amount of the modified BoNT/E He or the polypeptide or the pharmaceutical composition to a subject to thereby treat the condition, wherein the condition is chosen from the group comprising 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
• Fig. 1 shows the structure of a botulinum neurotoxin, which consist of a light chain (LC) and heavy chain (HC).
• the heavy chain can be divided into two functional domains, the translocation domain (HN) and binding domain (He).
• the crystal structures of both BoNT/A (PDB code: 3BTA) and BoNT/E (PDB code: 3FFZ) are shown in Fig. 1 and have previously been determined and reveal two distinct domain organisations (Lacy et al., 1998; Kumaran et al., 2009).
• Fig. 2 shows the crystal structure of BoNT/Al He in complex with a glycosylated fourth extracellular domain (LD4) of SV2C.
• LD4 glycosylated fourth extracellular domain
• Fig. 3 shows modified locations within the binding domain of native botulinum neurotoxin serotype E used to generate a modified BoNT/E with binding affinity for SV2C.
• the novel toxin has been called BoNT/E2C or E2C.
• Fig. 4 shows the protein sequence alignment of BoNT/E3 (Uniprot: A2I2S5) and the modified BoNT/E, E2C. Boxed sequences in the BoNT/E sequence have been replaced by sequences taken from BoNT/Al (Uniprot: P0DPI1) and grafted into BoNT/E3 (BoNT/E subtype E3) to create E2C (inserted sequences indicated with boxes in the E2C-sequence). The residue numbering is based on the full length sequence of E2C and BoNT/E.
• Fig. 5 shows the modified locations within the binding domain of native botulinum neurotoxin serotype E used to generate E2C, mapped onto the crystal structure of BoNT/E.
• PDB: 3FFZ Kiaran et al., 2009. Only the He part of the structure is shown.
• Fig. 6a shows the protein sequence alignment of BoNT/Al (Uniprot: P0DPI1) and E2C. Boxed sequences have been taken from BoNT/Al and grafted into BoNT/E3 to create E2C. The residue numbering is based on the full length sequence of E2C and the full-length sequence of BoNT/Al.
• Fig. 6b shows the protein sequence alignment of BoNT/Al (Uniprot: P0DPI1) and E2C. Boxed sequences have been taken from BoNT/Al and grafted into BoNT/E3 to create E2C. The residue numbering is based on the full-length sequence of E2C and the full-length sequence of BoNT/Al. Compared to Fig. 6a, the inserted sequences (boxed) have been trimmed to a minimum of amino acids needed for E2C to exhibit binding affinity towards the SV2C receptor.
• Fig. 7 is a chromatogram (A280 trace) from size-exclusion purification of Hc/E2C using a Superdex200 26/600 column. The fraction containing Hc/E2C is indicated.
• Fig. 8 shows a SDS-PAGE analysis of purified Hc/E2C
• 'M' denotes the lane containing molecular weight markers.
• Fig. 9 shows (A) Design of N-terminal 6x histidine-tagged and Flag-tagged constructs for a SV2 binding assay. 1: indicates a histidine-tag; 2: indicates a flag-tag; 3: indicates a Tobacco Etch Virus protease (TEV) site. Arrow indicates the cleaved bond upon incubation with TEV protease.
• B Schematic diagram of the SV2-binding ELISA. 4: indicates immobilised GST- tagged SV2 (only the fourth extracellular domain of SV2) on plate; 5: indicates binding of tagged Hcto SV2, and 6: indicates detection of bound He with anti-flag HRP (Horseradish peroxidase)-conjugated antibody.
• Fig. 10 shows binding of FLAG-tagged Hc/Al ( ⁇ ), Hc/El ( ⁇ ), and Hc/E2C ( A) to immobilised GST-tagged SV2C (only the fourth extracellular domain of SV2C) measured using an ELISA. Data were fit using a four-parameter logistic regression.
• Figs 11a and lib show protein sequence alignment of BoNT/E subtypes with E2C modifications to enhance SV2C binding.
• E2C modifications are boxed above the sequences and positions for substitutions are marked with a box in each subtype E1-E12.
• Fig. 12 shows extended use of Hc/E2C.
• the Hc/E2C domain can be fused onto the LC and H N domains of any other BoNT serotype or BoNT-like protein.
• the Hc/E2C can be fused to any other functional domain of interest.
• BoNT Botulinum neurotoxin encompasses any polypeptide or fragment from a Botulinum neurotoxin.
• the term BoNT may refer to a full-length BoNT.
• BoNT may refer to a fragment of the BoNT that can execute the overall cellular mechanism whereby a BoNT enters a neuron and inhibits neurotransmitter release.
• BoNT may simply refer to a fragment of the BoNT, without requiring the fragment to have any specific function or activity.
• translocation domain means a BoNT domain that can execute the translocation step of the intoxication process that mediates BoNT light chain translocation.
• an H N facilitates the movement of a BoNT light chain across a membrane into the cytoplasm of a cell.
• binding domain is synonymous with "He 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.
• nucleic acid and “gene” are used interchangeably to describe a nucleotide sequence, or a polynucleotide, encoding for a polypeptide.
• botulinum neurotoxins comprise a light chain (LC), linked by a single disulphide bridge to the heavy chain (HC).
• the Heavy chain (HC) holds two of the functional domains, with the N-terminal translocation domain (HN) and the C-terminal binding domain (He), while LC is responsible for intracellular catalytic activity.
• the He thus comprises the receptor binding domains, which are able to specifically and irreversibly bind to the specific receptors expressed on susceptible neurons, whereas the HN forms a channel that allows the attached LC to translocate from endosomal-like membrane vesicles into the cytosol.
• BoNTs botulinum neurotoxins
• BoNT/E botulinum neurotoxins
• BoNT/E only targets a subset of the available receptors found on neuronal cells (SV2A and SV2B) compared with BoNT/A, which can utilise all isoforms (SV2A, SV2B, SV2C).
• BoNT/E may have a faster onset rate than BoNT/A but it is unable to utilise all of the available SV2 receptor isoforms, which may potentially reduce its potency.
• the chimeras formed by Wang et al. do not take into account the potential interaction between the He and the H N domains, which may be important for overall toxin stability and during toxin translocation.
• the ganglioside- binding site of the BoNT/E may also be modified.
• it would be desirable with a BoNT chimera that ensures that any potential interface between the Hcand H N domains are maintained and that the ganglioside-binding site from BoNT/E is maintained.
• BoNT/E-like chimera toxin based on the native sequences of BoNT/A and BoNT/E and its production.
• the BoNT/E-like chimera toxin being produced via an advanced three-dimensional structural design process, which has resulted in a BoNT/E-like toxin that recognises SV2C and binds with greater affinity to SV2C than native BoNT/E
• the novel toxin has been called BoNT/E2C or E2C.
• the Hc/E2C construct comprises residues 853 to 1259 of the full-length BoNT/E2C sequence.
• BoNT/E2C R348A/Y351F see supplementary information for protein sequence and DNA sequence, SEQ ID NOs: 5 and 12 (including a tag sequence, aa residue 1-26)) (Agarwal, Binz & Swaminathan, 2005).
• SV2C cDNA encoding human SV2C residues 473 to 567
• pGEX-5X-l vector GenScript, NJ, USA.
• the construct comprises an N-terminal glutathione S-transferase (GST)-tag, FactorXa protease cleavage site, and SV2C 473 to 567 (GST-SV2C, see supplementary information for amino acid and DNA sequence, SEQ ID NOs: 7 and 11).
• Each construct was transformed into BL21 (DE3) E. coli (New England BioLabs, USA). An identical expression protocol was used for all proteins. Transformed cells were grown in terrific broth medium containing 50 pg/ml kanamycin (for Hc/Al,Hc/E3,Hc/E2C) or 100 pg/ml ampicillin (GST-SV2C) in a LEX bioreactor system (Epyphite3, Canada). Cells were grown at 37°C until they reached an OD600 of 0.6. The temperature was reduced to 16°C and protein expression induced at an OD600 of 1.0 by the addition of IPTG to a final concentration of 1 mM. After induction cells were allowed to continue to grow for a further 18 hours. Cells were harvested by centrifugation at 6000 x g for 15 min.
• Hc/E2C and Hc/E3 were purified using the same method.
• the cell paste was resuspended in 50 mM HEPES, 200 mM NaCI, 25 mM Imidazole, pH 7.4.
• Cells were lysed with a single pass through an Emulsiflex C3 cell disruptor (Avestin) at 20 kPSI. Lysate was cleared by ultracentrifugation at 158,000 x g for 30 min.
• Clarified lysate was loaded onto a 5 ml HisTrap HP column (GE Healthcare) and Hc/E2C or Hc/E3 was eluted using 50 mM HEPES, 200 mM NaCI, 500 mM Imidazole, pH 7.4.
• the eluted protein was further purified by size-exclusion chromatography (Superdex200 26/600, GE Healthcare) in 50 mM HEPES, 200 mM NaCI, pH 7.4.
• the cell paste was resuspended in 100 mM HEPES, 500 mM NaCI, 10 mM imidazole, 10% glycerol, 0.5 mM TCEP, pH 8.0 and lysed by sonication. Lysate was cleared by centrifugation at 49,000 x g for 20 min before filtration though a 0.45 pm filter. Clarified lysate was loaded onto a 5 ml HisTrap HP column (GE Healthcare) and eluted using 20 mM HEPES, 500 mM NaCI, 500 mM imidazole, 10% glycerol, 0.5 mM TCEP, pH 7.5.
• the eluted protein was further purified by size-exclusion chromatography (Superdex200 16/600, GE Healthcare) in 20 mM HEPES, 300 mM NaCI, 10% glycerol, 0.5 mM TCEP, pH 7.5.
• the cell paste was resuspended in phosphate-buffered saline (PBS, Sigma P4417) and lysed by a single pass through an Emulsiflex C3 cell disruptor (Avestin) at 20 kPSI. Lysate was cleared by ultracentrifugation at 158,000 x g for 30 min. Clarified lysate was loaded onto a 5 ml GST rap 4B column (GE Healthcare) and GST-SV2C was eluted using 50 mM Tris-HCI, 150 mM NaCI, 10 mM reduced glutathione, pH 8.0. The eluted protein was further purified by size- exclusion chromatography (Superdex200 16/600, GE Healthcare) in 50 mM Tris-HCI, 100 mM NaCI, pH 8.0.
• PBS phosphate-buffered saline
• Avestin Emulsiflex C3 cell disruptor
• 96-well plates (Nunc-lmmunoTM MicroWellTM 96-well solid plates) were coated with GST-SV2C (10 pg/ml in 100 mM Tris, pH 8.0) by the addition of 100 pi per well and incubation at 4°C for 16 hours. Wells were washed three times with PBS-T/0.1% BSA (200 mI phosphate buffered saline, 0.05% [v/v] Tween-20, 0.1% [w/v] bovine serum albumin). Non-specific binding-sites were blocked by incubation with 200 mI PBS-T/2% BSA for 1 hour at 22°C.
• PBS-T/0.1% BSA 200 mI phosphate buffered saline, 0.05% [v/v] Tween-20, 0.1% [w/v] bovine serum albumin
• He domains were added to the wells (serial dilution with concentration range of 10 mM to 500 pM for each sample in triplicate). He domains were left to incubate at 22°C for 1 hour before the wells were washed with 100 mI PBS/0.1% BSA three times.
• the detection antibody (Monoclonal ANTI-FLAG ® M2-Peroxidase (HRP) antibody,
• BoNT/A subtypes are known to utilise all three isoforms of SV2 and it is likely that each isoform shares a common binding mode.
• BoNT/E2C or E2C which consisted of a BoNT/E sequence which was modified to contain parts of BoNT/A
• BoNT/Al He Hc/Al
• Fig. 2 The crystal structures of BoNT/Al He (Hc/Al) in complex with both non-glycosylated and glycosylated SV2C, Fig. 2, have been previously published and released into the PDB as 4JRA and 5JLV, respectively (Benoit et al., 2014; Yao et al., 2016). These structures along with their associated publications highlight some of the interactions between BoNT/Al and SV2C, which we could investigate for use in the design of E2C.
• the fourth luminal domain of SV2C (residues 459 to 578, Uniprot: Q496J9) was found to form mostly backbone-backbone interactions with a beta-strand from the BoNT/Al He (residues 1141 to 1146).
• R1156 of BoNT/Al forms a cation-pi-stacking interaction with F563 of SV2C. This binding interface is then further complemented by interactions to an N-linked glycan from SV2 (Yao et al., 2016).
• BoNT/E Proteins from the BoNT/E serotypes differ from BoNT/A as they cannot utilise SV2C but are known to interact with SV2A and SV2B (Dong et al., 2008). However, there exists no structural information regarding the BoNT/E-SV2A/B complex and it is likely to differ from that of BoNT/A. Structures of BoNT/E alone have been determined which were used for analysis instead.
• BoNT/E A full-length crystal structure of BoNT/E is available in the PDB as 3FFZ (Kumaran et al., 2009), see Fig. 1.
• the sequences of Hc/Al and Hc/E3 were also aligned using ClustalO (Sievers et al., 2011) and their crystal structures aligned using PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrodinger, LLC). The He sequences share only 50% amino acid identity, while their core structural fold is conserved.
• BoNT/E BoNT/El - /E12 subtypes share between 88% and 99% amino acid sequence identity it is highly likely that the modifications used to modify the native BoNT/E3 sequence could be applied to all other BoNT/E subtypes (E1-E12) in order to generate the same effect as with BoNT/E3.
• Substitution 1 is an optional modification, and corresponds to amino acids 917-922 of the full-length sequence BoNT/Al: FNLESS (Phe Asn Leu Glu Ser Ser). These amino acids may replace amino acids starting at position 891-896 in the full-length native/non-modified BoNT/E sequence (Figs 6a, 6b).
• Substitution 2a is a non-optional modification and corresponds to amino acid 951-958 of the full-length sequence of BoNT/Al: KYFNSISL (Lys Tyr Phe Asn Ser lie Ser Leu). These amino acids may replace amino acids at positions 925-933in the full-length non-modified BoNT/E sequence (Fig. 6a). Substitution 2a may be trimmed to substitution 2b and corresponds to amino acid 951-957 of the full-length sequence of BoNT/Al: KYFNSIS (Lys Tyr Phe Asn Ser lie Ser). These amino acids may replace amino acids at positions 925-932 in the full-length non-modified BoNT/E sequence (Fig. 6b).
• Substitution 3 is a non-optional modification and corresponds to amino acid 980-981 of the full-length sequence of BoNT/Al: YG (Tyr Gly). These amino acids replace amino acids 956- 957 in the non-modified BoNT/E full-length sequence (Figs 6a and 6b).
• Substitution 4a is a non-optional modification and corresponds to amino acids 1002- 1006 of the full-length sequence of BoNT/Al: SQMIN (Ser Gin Met lie Asn). These amino acids replace amino acids 978-982 in the non-modified BoNT/E full-length sequence (Fig. 6a). Substitution 4a may be trimmed to substitution 4b and corresponds to amino acids 1004-1005 of the full-length sequence of BoNT/Al: Ml (Met lie). These amino acids may replace amino acids 980-981 in the full-length non-modified BoNT/E sequence (Fig. 6b).
• Substitution 5a is a non-optional modification and corresponds to amino acid 1057- 1064 of the full-length sequence of BoNT/Al: LDGCRDTH (Leu Asp Gly Cys Arg Asp Thr His). These amino acids may replace amino acids 1033-1039 in the full-length non-modified BoNT/E sequence (Figs 6a). Substitution 5a can be trimmed to substitutions 5bl and 5b2 and correspond to amino acids 1059 (G (Gly)) and 1061-1064 (RDTH (Arg Asp Thr His)) of the full- length sequence of BoNT/Al, respectively. These amino acids may replace amino acid 1035 and 1037-1039, respectively, in the full-length non-modified BoNT/E sequence (Fig. 6b).
• Substitution 6 is an optional modification and corresponds to amino acid 1122 of the full-length sequence of BoNT/Al: Y (Tyr).
• the amino acid may replace the amino acid at position 1097 of the full-length non-modified BoNT/E sequence (Figs 6a and 6b).
• Substitution 7a is a non-optional modification and corresponds to amino acids 1137- 1156 of the full-length sequence of BoNT/Al: KGPRGSVMTTNIYLNSSLYR (Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn lie Tyr Leu Asn Ser Ser Leu Tyr Arg).
• the amino acids may replace the amino acids 1109-1123 of the full-length non-modified BoNT/E sequence.
• Substitution 7a can be trimmed to substitutions 7bl, 7b2, 7b3 and 7b4 and correspond to amino acids 1138-1139 (GP (Gly Pro)), amino acid 1141 (G (Gly)), amino acid 1144-1151 (MTTNIYLNSS (Met Thr Thr Asn lie Tyr Leu Asn Ser Ser)), and 1156 (R (Arg)) of the full-length BoNT/Al sequence.
• the amino acids replacing the amino acids 1110-1111, 1113, 1115-1120 and 1123, respectively, of the full-length non-modified BoNT/E sequence.
• Substitution 8a is a non-optional modification and corresponds to amino acids 1286- 1296 of the full-length sequence of BoNT/Al: PVDDGWGERPL (Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu).
• the amino acids may replace the amino acids 1244-1252 of the full-length non- modified BoNT/E sequence.
• Substitution 8a can be trimmed to substitutions 8bl, 8b2, 8b3 and 8b4, which correspond to amino acid 1287 (V (Val)), amino acid 1289 (D (Asp)), amino acid 1292 (G (Gly), and amino acid 1294-1296 (RPL (Arg Pro Leu)) of the full-length BoNT/Al sequence.
• the amino acids replacing amino acids 1245, 1247, 1250 and 1252, respectively, of the full-length non-modified BoNT/E sequence.
• BoNT/E2C Using all eight substitutions, the sequence of BoNT/E2C alternates on 15 occasions between a native BoNT/Al or BoNT/E3 sequence (Figs 4,6a, 11a, lib) and also includes many novel and non-natural intra-molecular interfaces. None of the substitutions made to the native BoNT/E3 sequence are in the vicinity of the conserved ganglioside-binding site and thus we are confident that the interaction with the native ganglioside receptor remains intact. The resulting molecule named BoNT/E2C or E2C should be able to bind SV2C.
• Figs 4 and 6 BoNT/E2C and BoNT/E2C_R348A_Y351F, see supplementary information for protein and DNA sequence, SEQ ID NOs: 5, 6, 12, 13 (shown with tag sequence, aa residue 1-26)).
• two of the eight substitutions, 1 and 6, are optional substitutions. Including all eight substitutions may result in an E2C binding with higher affinity to the SV2C receptor than an E2C comprising only the six non-optional substitutions.
• Hc/E2C which encompasses all of the sequence modifications and includes all of the receptor-binding sites, by recombinant expression in E. coli.
• the expressed protein was soluble and purified by affinity chromatography using Ni-sepharose resin followed by size-exclusion chromatography (SEC).
• SEC size-exclusion chromatography
• the SEC produced a single distinct peak for Hc/E2C at the predicted size, demonstrating the viability of the engineered construct (Fig. 7).
• Purified Hc/E2C was analysed by SDS-PAGE and determined to be > 95% pure from inspection (Fig. 8).
• Hc/E2C has a greater affinity toward non-glycosylated SV2C than Hc/Al
• ELISA enzyme-linked immunosorbent assay
• the GST-SV2C protein then was adsorbed onto the surface of 96-well immunoassay plates and the binding of Hc/E2C, Hc/Al, and Hc/E3 was measured at different protein concentrations.
• Previously published literature has established that BoNT/E3 is unable to bind with high-affinity to SV2C while BoNT/Al is capable of binding both glycosylated and non-glycosylated forms of SV2C (Rummel et al., 2009; Mahrhold et al., 2013; Yao et al., 2016).
• Hc/Al and Hc/E2C displayed strong binding to SV2C (Kd values of 85 nM and 54 nM, respectively) while Hc/E3 showed very weak affinity in comparison (approximate Kd 2.6 mM) (Fig. 10). Due to the weak affinity of Hc/E3, the fit could not be described with a high confidence.
• ELISA data clearly demonstrate the ability for E2C to bind SV2C with an affinity higher than Hc/Al indicating that the design of E2C was successful.
• BoNT/E (BoNT/El - /E12) subtypes share between 88% and 99% amino acid sequence identity. It is therefore likely that the modifications used to generate E2C could be applied to all other BoNT/E subtypes (E1-E12) in order to generate the same effect as with BoNT/E3 discussed above. A sequence alignment of all BoNT/E subtypes was performed and the location of E2C modification is indicated (Fig. 11a and Fig. lib). We suggest that production of any of these BoNT/E subtypes (or similar sequences) with the provided changes would result in a similar effect as E2C based on subtype 3.
• BoNT/E2C an engineered BoNT/E protein which is capable of binding to the SV2C protein receptor.
• the engineered E2C He domain could also be used independently to either generate novel chimeric toxins, that contain a native Lcand HN sequence fused to the engineered Hc/E2C, or generate novel proteins that contain other functional domains fused to Hc/E2C (Fig. 12).
• Chimeric BoNTs containing Hc/E2C may show an increased affinity to SV2 isoforms, particularly SV2C, and also an increased potency compared with the native BoNT. Novel proteins containing Hc/E2C will allow targeting of functional proteins to cells expressing SV2C.
• BoNT/E2C is a BoNT/E-like toxin which has been engineered to recognise the SV2C protein. This novel design is of great potential and could prove more efficacious than current commercial BoNT formulations. Most commercial BoNT formulations are based on the BoNT/Al subtype with an onset time of approximately 2 - 3 days. Decreasing this time would be highly beneficial for novel therapeutics. There exists a natural toxin serotype, BoNT/E, which has been shown to have a fast onset time. However, it is likely to be less potent than BoNT/Al as it can only utilise a subset of the available protein receptors. The main innovation in BoNT/E2C is the He domain design, which may also be used in combination with other functional domains from proteins such as BoNTs.
• Benoit RM Frey D, Hilbert M, Kevenaar JT, Wieser MM, Stirnimann CU, McMillan D, Ceska T, Lebon F, Jaussi R, Steinmetz MO, Schertler GFX, Hoogenraad CC, Capitani G, Kammerer RA. 2014. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin
• Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors. PLoS pathogens 7:el002008.
• Botulinum neurotoxins C, e and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor.
• N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin

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