# Patent

## Title
Genetically engineered clostridial genes, proteins encoded by the engineered genes, and uses thereof

## Abstract

The present invention relates to an isolated Clostridial neurotoxin propeptide having a light chain region, a heavy chain region, where the light and heavy chain regions are linked by a disulfide bond, and an intermediate region connecting the light and heavy chain regions. An isolated nucleic acid molecule encoding a Clostridial neurotoxin propeptide is also disclosed. Also disclosed is an isolated, physiologically active Clostridial neurotoxin produced by cleaving a Clostridial neurotoxin propeptide, a vaccine or antidote thereof, and methods of immunizing against or treating for toxic effects of Clostridial neurotoxins. Methods of expressing recombinant physiologically active Clostridial neurotoxins are also disclosed. Also disclosed is a chimeric protein having a heavy chain region of a Clostridial neurotoxin and a protein with therapeutic functionality. A treatment method is also disclosed.

## Background
This application is a division of U.S. patent application Ser. No. 12/762,909, filed Apr. 19, 2010, which is a division of U.S. patent application Ser. No. 11/284,930, filed Nov. 22, 2005, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 60/630,175, filed Nov. 22, 2004, which are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
This invention relates to isolated Clostridial propeptides and neurotoxins, vaccines or antidotes thereof, methods of immunizing and treating subjects, isolated nucleic acid molecules encoding Clostridial propeptides and neurotoxins, methods of expression, chimeric proteins, and treatment methods.
BACKGROUND OF THE INVENTION
The Clostridial neurotoxins are a family of structurally similar proteins that target the neuronal machinery for synaptic vesicle exocytosis. Produced by anaerobic bacteria of the Clostridium genus, botulinum neurotoxins (“BoNT”s, seven immunologically distinct subtypes, A-G) and Tetanus neurotoxin (“TeNT”) are the most poisonous substances known on a per-weight basis, with an LD50 in the range of 0.5-2.5 ng/kg when administered by intravenous or intramuscular routes (National Institute of Occupational Safety and Health, “Registry of Toxic Effects of Chemical Substances (R-TECS),” Cincinnati, Ohio: National Institute of Occupational Safety and Health (1996)). BoNTs target cholinergic nerves at their neuromuscular junction, inhibiting acetylcholine release and causing peripheral neuromuscular blockade (Simpson, “Identification of the Major Steps in Botulinum Toxin Action,” Annu. Rev. Pharmacol. Toxicol. 44:167-193 (2004)). BoNT serotypes A, B, and E are considered to represent the most significant threat to military and civilian populations, particularly because they can be aerosolized and delivered by inhalation (Amon et al., “Botulinum Toxin as a Biological Weapon: Medical and Public Health Management,” JAMA 285:1059-1070 (2001)).
Though much work has been done to develop vaccines or antidotes which are effective against poisoning with Clostridial neurotoxins, the effectiveness of available products is limited because the available inactivated toxin preparations do not optimally mimic the native toxin. No therapeutic antidotes or vaccines have been approved for widespread use, though some preparations are available for limited use under specific circumstances. The NIAID Biodefense Research Agenda has identified the development of countermeasures against Clostridial neurotoxins as one of its most pressing goals (National Institute of Allergy and Infectious Diseases, “NIAID Biodefence Research Agenda for CDC category A Agents” NIH Publication #03-5308 (2002)). A prime target is understanding and preventing neurotoxin entry into target cells Immunological approaches have utilized passive protection via injection of antibodies as antitoxins, or active immunization via vaccination with toxoids, toxins chemically or genetically transformed to render them non-toxic but still immunogenic (Ramon et al., “Sur L'immunization Antitetanique et sur la Production de L'antitoxine Tetanique,” Compt. Rend. Soc. Biol. 93:508-598 (1925)). Antibody-based anti-toxins are available in limited quantities, but no protective vaccine against Clostridial neurotoxins has been approved. A pentavalent botulinum toxoid (ABCDE), consisting of toxins inactivated by temperature or cross-linked with formaldehyde, is available in limited quantities, and has been shown to induce antibodies in laboratory workers and military personnel (National Institute of Allergy and Infectious Diseases, “NIAID Biodefence Research Agenda for CDC category A Agents. Progress Report,” NIH Publication #03-5435 (2003)). An inactivated heavy chain toxoid administered by inhalation was found to protect animals against inhaled toxin doses 104 times the LD50 (Park et al., “Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component,” Infect. Immun. 71:1147-1154 (2003)). An investigational heptavalent antitoxin (A-G reactive, equine origin) against BoNT is being developed by the U.S. Department of Defense and is now being tested. Initial data demonstrate the general safety of this antitoxin, though it displays some cross-species reactogenicity in humans. Another investigational BoNT anti-toxin is based on a combination of three recombinant monoclonal antibodies, which neutralize BoNT A with a high potency (Nowakowski et al., “Potent Neutralization of Botulinum Neurotoxin by Recombinant Oligoclonal Antibody,” Proc. Natl. Acad. Sci. USA 99:11346-11350 (2002)). Development and testing of human monoclonal antibodies to BoNT B-G is also currently in progress and supported by NIAID (National Institute of Allergy and Infectious Diseases, “NIAID Biodefence Research Agenda for CDC category A Agents. Progress Report,” NIH Publication #03-5435 (2003)).
Several laboratories are attempting to develop recombinant Clostridial toxin genes or fragments thereof The Department of Defense has developed a vaccine based on expression of the receptor-binding domain of the BoNT A heavy chain (National Institute of Allergy and Infectious Diseases, “NIAID Biodefence Research Agenda for CDC Category A Agents. Progress Report,” NIH Publication #03-5435 (2003); Byrne et al., “Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate,” Infect. Immun. 66:4817-4822 (1998); and Pless et al., “High-Affinity, Protective Antibodies to the Binding Domain of Botulinum Neurotoxin Type A,” Infect. Immun. 69:570-574 (2001)). A similar approach with a recombinant BoNT F fragment expressed in Salmonella typhimurium was found to provide partial protection of animals against the toxin (Foynes et al., “Vaccination Against Type F Botulinum Toxin Using Attenuated Salmonella enterica var Typhimurium Strains Expressing the BoNT/F HC Fragment,” Vaccine 21:1052-1059 (2003)). A catalytically active non-toxic derivative of BoNT A expressed in E. coli was reported to induce toxin-neutralizing antibodies and protect animals from a BoNT challenge (Chaddock et al., “Expression and Purification of Catalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridium botulinum Toxin Type A,” Protein Expr. Purif. 25:219-228 (2002)). A catalytically inactive, full-length derivative of BoNT C expressed in E. coli was immunogenic in mice, though limitations of this system hinder expression of full-length native and active recombinant toxin (Kiyatkin et al., “Induction of an Immune Response by Oral Administration of Recombinant Botulinum Toxin,” Infect. Immun. 65:4586-4591 (1997)). Rummel et al. (“Synaptotagmins I and II Act as Nerve Cell Receptors for Botulinum Neurotoxin G,” J. Biol. Chem. 279:30865-30870 (2004) (“Rummel I”)) and Rummel et al. (“The Hcc-domain of Botulinum Neurotoxins A and B Exhibit a Singular Ganglioside Binding Site Displaying Serotype-Specific Carbohydrate Interaction,” Mol. Microbiol. 51:631-643 (2004) (“Rummel II”), report full-length BoNT A, B, and G neurotoxins expressed in an E. coli from plasmids encoding the respective full-length genes. Rummel I and Rummel II also report several derivatives of BoNT genes. The neurotoxins described in Rummel I and Rummel II are active only at very high concentrations. This is likely due to the fact that the neurotoxins expressed by Rummel I and Rummel II are denatured during expression, extraction, and purification from E. coli and achieve low physiological activity of the single chain BoNT propeptide due to improper disulfide bonding. Thus, although Rummel I and Rummel II may in fact have produced full-length recombinant BoNT peptides of serotypes A, B, and G, the properties of the neurotoxins described do not possess native structures and physiological activity.
The widely used E. coli expression system may be problematic for some proteins, because the E. coli cytosol may not provide the non-reducing environment needed for maintenance of disulfide bridges critical to the native toxin structure (Alberts et al., Molecular Biology of the Cell, Third Edition, Garland Publishing Inc., 112, 113, 488, 589). In addition, E. coli based expression systems also present practical problems associated with endotoxin removal. These limitations emphasize the importance of selecting an expression system capable of producing recombinant molecules that retain the native toxin structure and biological activity.
Data from multiple laboratories suggest that the C-terminal moiety of Clostridial toxin heavy chains (“Hc”), or the intact heavy chain (“HC”) expressed or prepared by reduction/denaturation from native toxins, are functionally altered and therefore require a  ̃10,000-fold molar excess to delay the onset of toxin-induced paralysis (Li et al., “Recombinant Forms of Tetanus Toxin Engineered for Examining and Exploiting Neuronal Trafficking Pathways,” J. Biol. Chem. 276:31394-31401 (2001); Lalli et al., “Functional Characterization of Tetanus and Botulinum Neurotoxins Binding Domains,” J. Cell Sci. 112:2715-2724 (1999)). Some of these preparations have been completely inactive in this assay (Daniels-Holgate et al., “Productive and Non-Productive Binding of Botulinum Neurotoxin A to Motor Nerve Endings are Distinguished by Its Heavy Chain,” J. Neurosci. Res. 44:263-271 (1996)). The low efficiency of HC and Hc may be due to either their increased binding affinity to non-productive sites on cells normally mediating toxin trafficking or their conformational differences from the native toxin which results in a low binding affinity for the specific binding sites at the target cells. In either case, incorrect folding, altered post-translational modification, a requirement for the N-terminal portion of the molecule (Koriazova et al., “Translocation of Botulinum Neurotoxin Light Chain Protease through the Heavy Chain Channel,” Nat. Struct. Biol. 10:13-18 (2003)), or multiple other changes, may be responsible for these functionally important deficiencies. These facts suggest that the currently available preparations of BoNT or its derivatives are poor mimics of the native toxin, which may limit their therapeutic potential.
The methods currently available to produce inactivated derivatives of BoNTs as vaccines or antidotes to BoNT poisoning have met with limited success. This can be due to several factors. First, the methods used to inactivate BoNT prepared from Clostridial cultures are harsh, and may alter the toxin's native conformation in ways that may influence its immunogenicity or trafficking and absorption. Second, methods based on producing recombinant toxins have thus far only succeeded in producing either inactive toxin molecules or fragments of its protein domains. In both cases, the recombinant molecules produced are by definition significantly different from native toxin, particularly with respect to post-translational processing and disulfide bonding. Though inactivated toxins and toxin fragments have been shown to be immunogenic, the pool of polyclonal antibodies they generate will include a fraction recognizing epitopes present only on misfolded toxins.
Another area in which Clostridial neurotoxins have been extensively studied relates to their clinical use to treat dystonias, and to temporarily correct aesthetic defects in skin. These indications are specific to the neurotoxins produced by strains of Clostridium botulinum (BoTox), because they can be used at extremely small doses to locally paralyze specific muscles and thereby achieve therapeutic goals. All of the current products used for this indication are produced from Clostridial cultures, and there have been no reports of an active BoTox molecule produced using any type of genetic engineering technology.
A further area of interest is derived from the ability of Clostridial neurotoxins to pass undegraded through epithelial barriers via transcytosis, and specifically target nervous tissue. This has led to suggestions that Clostridial neurotoxins can be used to enable oral and inhalational carriers for therapeutic agents that cannot normally be delivered via these routes of administration, and delivery vehicles which can specifically target the peripheral and central nervous system.
The present invention is directed to overcoming these and other limitations in the art.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to an isolated Clostridial neurotoxin propeptide. The propeptide has a light chain region, a heavy chain region, where the light and heavy chain regions are linked by a disulfide bond, and an intermediate region connecting the light and heavy chain regions. The intermediate region has a highly specific protease cleavage site which has three or more specific adjacent amino acid residues that are recognized by the highly specific protease in order to enable cleavage.
Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the above Clostridial neurotoxin propeptide as well as expression systems and host cells containing this nucleic acid molecule.
A further aspect of the present invention relates to an isolated, physiologically active Clostridial neurotoxin produced by cleaving the above Clostridial neurotoxin propeptide. The propeptide is cleaved at the highly specific protease cleavage site. The light and heavy chain regions are linked by a disulfide bond.
Yet another aspect of the present invention relates to a vaccine or antidote including the above physiologically active, atoxic, Clostridial neurotoxin produced by cleaving the isolated Clostridial neurotoxin propeptide at the highly specific protease cleavage site. The light and heavy chain regions are linked by a disulfide bond.
Still another aspect of the present invention relates to method of immunizing a subject against toxic effects of a Clostridial neurotoxin. This method involves administering the above vaccine to the subject under conditions effective to immunize the subject against toxic effects of Clostridial neurotoxin.
Yet a further aspect of the present invention relates to a method of treating a subject for toxic effects of a Clostridial neurotoxin. This method involves administering an antidote comprising the above physiologically active, atoxic, Clostridial neurotoxin produced by cleaving the isolated Clostridial neurotoxin propeptide under conditions effective to treat the subject for toxic effects of Clostridial neurotoxin.
Still a further aspect of the present invention relates to a chimeric protein including a first protein or protein fragment having a heavy chain region of a Clostridial neurotoxin and a second protein or protein fragment linked to the first protein or protein fragment.
Another aspect of the present invention relates to a method of expressing a recombinant physiologically active Clostridial neurotoxin. This method involves providing a nucleic acid construct having a nucleic acid molecule encoding an isolated Clostridial neurotoxin propeptide. The nucleic acid construct has a heterologous promoter operably linked to the nucleic acid molecule and a 3′ regulatory region operably linked to the nucleic acid molecule. The nucleic acid construct is introduced into a host cell under conditions effective to express the physiologically active Clostridial neurotoxin.
A further aspect of the present invention relates to a treatment method. This method involves contacting a patient with an isolated, physiologically active, toxic, Clostridial neurotoxin produced by cleaving the above isolated Clostridial neurotoxin propeptide.
The present invention relates to a genetic engineering platform that enables rationale design of therapeutic agents based on Clostridial toxin genes. The genetic engineering scheme is based on a two-step approach. For each Clostridial toxin serotype, gene constructs, expression systems, and purification schemes are designed that produce physiologically active, recombinant Clostridial neurotoxin. This ensures that the recombinant toxin derivatives retain structural features important for developing therapeutic candidates, or useful biologic reagents. Using the genetic constructs and expression systems developed by this paradigm, selective point mutations are then introduced to create atoxic recombinant derivatives. This two-step approach is designed to ensure that the recombinant toxin derivatives retain the immunogenicity, absorption profile, and trafficking pathways of native toxin, allowing the atoxic derivatives to have optimized therapeutic and biological properties. They also enable useful chimeric proteins to be created.
Genetically engineered forms of recombinant toxins which structurally and functionally mimic native toxins are superior to the toxoids currently in development for therapeutic purposes. They provide new approaches which can produce customized toxin derivatives in large quantities, and with mutations specifically targeted to the creation of vaccines and toxin antidotes. By focusing on solving the problems associated with producing recombinant toxins, which are physiologically active, the inactivated toxin derivatives of the present invention have distinct advantages over currently available alternatives. This is particularly true with respect to their immunogenic activity and their ability to compete with native toxin for cellular binding sites.
The methodology described herein has additional scientific and practical value because it provides a broad platform enabling facile manipulation and expression of Clostridial toxin genes. This will facilitate studies of the mechanism of Clostridial toxin action, their intracellular trafficking, and the factors responsible for their ability to transit through specific cell types without activation or toxic consequences. In addition, the BoNT constructs created can provide new tools for delivering specific reagents or drugs via oral or inhalation routes, or specifically into peripheral neurons, and enable their controlled activation at the site of intended action. Other approaches to engineer delivery tools based on chemically modified heavy chains from Clostridial neurotoxins have had limited success, possibly because the methods used to inactivate the toxin interfere with protein spatial structure (Goodnough et al., “Development of a Delivery Vehicle for Intracellular Transport of botulinum Neurotoxin Antagonists,” FEBS Lett. 513:163-168 (2002), which is hereby incorporated by reference in its entirety).



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-C show comparative alignment of amino acid sequences of the seven wildtype botulinum neurotoxin serotypes, including Clostridium botulinum serotype A (SEQ ID NO: 1), Clostridium botulinum serotype B (SEQ ID NO: 2), Clostridium botulinum serotype C (SEQ ID NO: 3), Clostridium botulinum serotype D (SEQ ID NO: 4), Clostridium botulinum serotype E (SEQ ID NO: 5), Clostridium botulinum serotype F (SEQ ID NO: 6), and Clostridium botulinum serotype G (SEQ ID NO: 7). Gaps have been introduced to maximize homology. Amino acids identical in ≧50% of compared sequences are shown in black boxes Amino acids constituting the active site of the catalytic domain of metalloprotease are marked by stars. Disulfide bridge between neurotoxin cysteine residues of the light and heavy chain are shown as a long horizontal bracket. The amino acid residues constituting the minimal catalytic domain of the light chain are hatched. The first amino acid of the C-terminal part of the protein heavy chain (N872 for BoNT A), constituting receptor-binding domain are shown with the arrow Amino acids, absent in the mature dichain BoNT A molecule along with the aligned amino acids of the other BoNT serotypes are boxed. The white arrow is positioned at the first amino acid of the neurotoxins' heavy chain.
FIGS. 2A-C show comparative alignment, using the Clustal Program, of amino acid sequences of the seven botulinum neurotoxin serotypes, including Clostridium botulinum serotype A (SEQ ID NO: 8), Clostridium botulinum serotype B (SEQ ID NO: 9), Clostridium botulinum serotype C (SEQ ID NO: 10), Clostridium botulinum serotype D (SEQ ID NO: 11), Clostridium botulinum serotype E (SEQ ID NO: 12), Clostridium botulinum serotype F (SEQ ID NO: 13), and Clostridium botulinum serotype G (SEQ ID NO: 14), which have been slightly modified in accordance with the present invention. Gaps have been introduced to maximize homology. Amino acids identical in ≧50% of compared sequences are shown in black boxes Amino acids constituting the active site of the catalytic domain of metalloprotease are marked by stars. Disulfide bridge between neurotoxin cysteine residues of the light and heavy chain are shown as a long horizontal bracket. The amino acid residues constituting the minimal catalytic domain of the light chain are hatched. The first amino acid of the C-terminal part of the protein heavy chain (N876 for BoNT A), constituting receptor-binding domain are shown with the arrow Amino acids, absent in the mature dichain BoNT A molecule along with the aligned amino acids of the other BoNT serotypes are boxed. The white arrow is positioned at the first amino acid of the neurotoxins' heavy chain. Amino acid residues are modified in comparison with the wild type sequence to restrict trypsin-like proteolysis Amino acids which constitute the insertion/modification into the wild type amino acid residues and represent an enterokinase cleavage site are also shown.
FIGS. 3A-B illustrate features of the wild type BoNT A protein and gene (wt), and its toxic recombinant derivative (td). FIG. 3A is a schematic representation of the native BoNT A (wt) dimer, illustrating the catalytic ( ̃50 kDa), translocation ( ̃50 kDa), and receptor-binding ( ̃50 kDa) domains. FIG. 3B is a comparison of the nucleotide (SEQ ID NO: 65 (wt) and SEQ ID NO: 66 (td)) and amino acid (SEQ ID NO: 1 (wt) and SEQ ID NO: 8 (td)) sequences of the native BoNT A (wt) and its recombinant toxic derivative (td), as generated in plasmid pLitBoNTA. Sequences common to both the wt and td genes are shown as black letters on a white background, or as white boxes. White letters on a black background represent the amino acids excised from the toxin propeptide to generate the mature wt toxin. The disulfide bonds joining the LC and HC are shown as long horizontal brackets. Grey letters indicate the unique endonuclease restriction sites introduced into non-coding portions of the td DNA sequence and the Shine-Dalgarno region of the wt sequence. All other mutations introduced to modify the construct properties are also shown in grey letters. The de novo enterokinase cleavage site inserted into the td propeptide is shown by an arrow. Amino acids proximal to conceived (wt) or executed (td) mutations are numbered.
FIGS. 4A-B show expression and purification of the toxic derivative of BoNT A (td) in E. coli. FIG. 4A shows 8% PAGE stained with Coomassie G-250. FIG. 4B shows a Western blot of the PAG shown in FIG. 4A, probed with polyclonal antibodies raised against the full-length BoNT A toxoid. Samples were treated with β-mercaptoethanol before separation. The protein molecular weight standards are shown to the far left. Lanes 1 and 2 are cleared lysate of E. coli transformed with pETcoco2 empty vector (Lane 1) or pETcocoBoNTA (Lane 2). Lane 3 is a purified preparation of native BoNT A used as positive control. Lane 4 and 5 are eluates from the Ni-NTA affinity purification of cleared E. coli lysates which have been transformed with pETcoco2 (Lane 4) or pETcocoBoNTA (Lane 5). SC: single chain propeptide. HC: Heavy Chain. LC: Light Chain.
FIG. 5 is a schematic representation of the three recombinant BoNT A derivatives expressed in a baculovirus system. BoNT A td: toxic derivative of BoNT A. BoNT A ad: atoxic derivative of BoNT A. BoNT A gfpd: green fluorescent protein (GFP) derivative of BoNT A. Further modifications introduced into the td sequence depicted in FIG. 3 include the introduction of a signal sequence and a hexahistidine tag (e.g., SEQ ID NO: 45) in front of the first native methionine for affinity purification. The difference between td and ad is a single amino acid substitution, E224>A, in the active center of toxin's catalytic domain. To create BoNT A gfpd, amino acids Tyr10-Leu416 of the native toxin's minimal catalytic domain were substituted with GFP. White and black arrows represent secretase and enterokinase cleavage sites, respectively.
FIG. 6 shows expression of BoNT A derivatives in a baculovirus system by Western blot, probed with polyclonal antibodies raised against full-length BoNT A toxoid. Samples were treated with β-mercaptoethanol before separation. Protein molecular weight standards are shown on the left. Lane 1, 2, 3, and 4: conditioned media from Sf9 cells infected with empty bacmid (Lane 1), or recombinant bacmids derived from pFBSBoNTA (Lane 2), pFBSBoNTAME224A (Lane 3) or pFBSGFPBoNTAHC (Lane 4). Lane 5 is native BoNT A as a positive control. Lanes 6, 7, 8, and 9: eluate after Ni-NTA affinity purification of conditioned media from Sf9 cells transfected with empty bacmid (Lane 6), or recombinant bacmids derived from pFBSBoNTA (Lane 7), pFBSBoNTAME224A (Lane 8), or pFBSGFPBONTAHC (Lane 9).
FIGS. 7A-B illustrate the concentration of recombinant enterokinase (rEK) required to effect complete cleavage of BoNT A toxic derivative (td) propeptide. FIG. 7A shows 8% PAGE stained with Coomassie G-250. FIG. 7B shows a Western blot of the gel in FIG. 7A, probed with polyclonal antibodies raised against full-length BoNT A toxioid. Samples were treated with (3-mercaptoethanol before the separation. Protein molecular weight standards are shown on the left. Different amounts of rEK were added to 1 μg of BoNT A td in rEK cleavage buffer and incubated at 20° C. for 8 hours. 10% of each reaction mixture was loaded per lane. The number of rEK units added per 1 g of BoNT A td were: no rEK added (Lane 1); 0.05 U of rEK (Lane 2); 0.1 U of rEK (Lane 3); 0.25 U of rEK (Lane 4); 0.5 U of rEK (Lane 5). Lane 6 is the positive control, with 0.1 μg of native BoNT A. The recombinant light chain is larger than the control because of construct design.
FIGS. 8A-D show selected features of the recombinant BoNT A derivatives illustrating their native disulfide bonding (FIGS. 8A and 8B), and the use of a signal sequence to increase secretion of the toxin derivative into the culture medium (FIGS. 8C and 8D). FIGS. 8A and 8B show PAGE of the indicated BoNT derivatives run on 10% PAGE gels, followed by Western blotting using polyclonal antibodies raised against full-length BoNT A toxioid. A protein molecular weight ladder is shown on the left. In FIG. 8A, the PAGE was run under non-reducing conditions before transfer to the nitrocellulose. In FIG. 8B, samples were treated with β-mercaptoethanol and run under reducing conditions before transfer to the nitrocellulose for Western blotting. Lane 1: Positive control, purified native BoNT A; Lane 2: BoNT A td cleaved with rEK; Lane 3: BoNT A ad cleaved with rEK; Lane 4: BoNT A gfpd cleaved with rEK. FIGS. 8C and 8D are fluorescent images of the adherent layer of Sf9 cells (2.105/cm2) in the SF 900 II medium at 12 hours post-infection (MOI ̃0.1) with recombinant baculovirus expressing BoNT A gfpd containing the signal peptide for secretion (FIG. 8C), or the control recombinant baculovirus expressing GFP without added signal peptide (FIG. 8D). Emission wavelength 508 nm, magnification factor ×200, exposure time 0.1 sec.
FIG. 9 is a BoNT A td purification table of 8% PAGE stained with Coomassie G-250. Samples were separated in the presence of β-mercaptoethanol. Lane 1: concentrated and dialyzed Sf9 medium, loaded on DEAE Sepharose; Lane 2: 100 mM NaCl eluate from DEAE Sepharose; Lane 3: 200 mM NaCl eluate from MonoS column; Lane 4: 60 mM imidazole eluate from Ni-NTA agarose; Lane 5: material, eluted from the FPLC gel-filtration column; Lane 6: material, eluted from the FPLC gel-filtration column and digested with rEK; Lane 7: positive control, purified native BoNT A. Protein molecular weight ladder is shown on the right.
FIGS. 10A-B illustrate a transcytosis assay for polarized cells. Human gut epithelial cells (T-84) or canine kidney cells (MDCK) will be grown subject to conditions that promote differentiation and polarization of the cell monolayer (FIG. 10A). An example of a polarized cell illustrating orientation of the apical membrane toward the top (accessible to medium in the insert) and the basal membrane oriented toward the bottom (accessible to medium in the well) (FIG. 10B). Cells will be grown on polycarbonate membranes coated with collagen in Transwell® porous bottom inserts. The inserts suspend the cell monolayer above the bottom of the well, enabling cells to feed from the top and the bottom, and to be exposed to toxin from the top and the bottom. Cultures grown in this manner differentiate into a polarized membrane with tight junctions.
FIGS. 11A-C illustrate the amino acid sequences of nine BoNT A chimeric proteins containing SNARE motif peptides substituted for alpha-helix domains in the light chain region aligned against the BoNT A ad protein (SEQ ID NO: 8). Chimera 1 (SEQ ID NO: 15) contains the full-length sequence of BoNT A ad with three SNARE motif peptides substituting light chain alpha-helix 1. Chimera 2 (SEQ ID NO: 16) contains the full-length sequence of BoNT A ad with two SNARE motif peptides substituting light chain alpha-helix 4. Chimera 3 (SEQ ID NO: 17) contains the full-length sequence of BoNT A ad with five SNARE motif peptides substituting light chain alpha-helices 1 and 4. Chimera 4 (SEQ ID NO: 18) contains the full-length sequence of BoNT A ad with three SNARE motif peptides substituting light chain alpha-helices 4 and 5. Chimera 5 (SEQ ID NO: 19) contains the full length sequence of BoNT A ad with six SNARE motif peptides substituting light chain alpha-helices 1, 4, and 5. Chimera 6 (SEQ ID NO: 20) contains the full length sequence of BoNT A ad with four SNARE motif peptides substituting light chain alpha-helices 4, 5, and 6. Chimera 7 (SEQ ID NO: 21) contains the full length sequence of BoNT A ad with five SNARE motif peptides substituting light chain alpha-helices 4, 5, 6, and 7. Chimera 8 (SEQ ID NO: 22) contains the full length sequence of BoNT A ad with seven SNARE motif peptides substituting light chain alpha-helices 1, 4, 5, and 6. Chimera 9 (SEQ ID NO: 23) contains the full length sequence of BoNT A ad with eight SNARE motif peptides substituting light chain alpha-helices 1, 4, 5, 6, and 7.



DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention relates to an isolated Clostridial neurotoxin propeptide. The propeptide has a light chain region, a heavy chain region, where the light and heavy chain regions are linked by a disulfide bond, and an intermediate region connecting the light and heavy chain regions. The intermediate region has a highly specific protease cleavage site which has three or more specific adjacent amino acid residues that are recognized by the highly specific protease in order to enable cleavage.
In a preferred embodiment, the isolated Clostridial neurotoxin propeptide is from Clostridium botulinum. Clostridium botulinum has multiple serotypes (A-G). Although the Clostridial neurotoxin propeptides of the present invention may be from any of the Clostridium botulinum serotypes, preferable serotypes are serotype A, serotype B, and serotype E.
Common structural features of the wild-type Clostridium botulinum neurotoxin propeptides are shown in FIG. 1. These structural features are illustrated using BoNT A propeptide as an example, and are generalized among all Clostridium botulinum serotypes. BoNT A propeptide has two chains, a light chain (“LC”) of Mr ̃50,000 and a heavy chain (“HC”) of Mr ̃100,000, linked by a disulfide bond between Cys429 and Cys453. As illustrated in FIG. 1, all seven BoNT serotype propeptides have a light chain region and a heavy chain region linked by a disulfide bond. Two essential Cys residues, one adjacent to the C-terminus of the light chain, and a second adjacent to the N-terminus of the heavy chain are present in all seven BoNT serotypes. These two Cys residues form the single disulfide bond holding the HC and LC polypeptides together in the mature neurotoxin. This disulfide bond enables the mature neurotoxin to accomplish its native physiological activities by permitting the HC and LC to carry out their respective biological roles in concert. The disulfide bond between HC and LC polypeptides in all seven serotypes is illustrated in FIG. 1 by the solid line joining the involved Cys residues. The outlined box in FIG. 1 illustrates the intermediate region defined by amino acid residues Lys438-Lys448 of BoNT A. This intermediate region identifies the amino acids eliminated during maturation of wild-type BoNT A, and believed to be excised by a protease endogenous to the host microorganism. This cleavage event, described infra, generates the biologically active BoNT HC-LC dimer. The outlined amino acid residues in FIG. 1, representing amino acid residues numbered approximately in the 420 to 450 range for all seven BoNT serotypes, can be considered as a region “non-essential” to the toxins' physiological activity and, therefore, represents targets for directed mutagenesis in all seven BoNT serotypes.
All seven BoNT serotypes contain Lys or Arg residues in the intermediate region defined by the box in FIG. 1 which make the propeptides susceptible to activation by trypsin. Native BoNT A propeptide recovered from young bacterial cultures can be activated by trypsinolysis, with production of intact, S—S bound light and heavy chain. Though multiple additional trypsin-susceptible sites are present in the propeptides, they are resistant to proteolysis due to their spatial positions within the native toxin molecule (Dekleva et al., “Nicking of Single Chain Clostridium botulinum Type A Neurotoxin by an Endogenous Protease,” Biochem. Biophys. Res. Commun. 162:767-772 (1989); Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998), which are hereby incorporated by reference in their entirety). A second site in the native propeptide of several BoNT serotypes can be susceptible to trypsin cleavage when subjected to higher enzyme concentrations or incubation times (Chaddock et al., “Expression and Purification of Catalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridium botulinum Toxin Type A,” Protein Expr. Purif. 25:219-228 (2002), which is hereby incorporated by reference in its entirety). This trypsin-susceptible site is located in the region adjacent to the toxin receptor binding domain. This region of the HC peptide is found to be exposed to solvent in BoNT serotypes for which information is available on their 3-D crystal structure (Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998); Swaminathan et al., “Structural Analysis of the Catalytic and Binding Sites of Clostridium botulinum Neurotoxin B,” Nat. Struct. Biol. 7:693-699 (2000), which are hereby incorporated by reference in their entirety).
In a preferred embodiment, the propeptide of the present invention has an intermediate region connecting the light and heavy chain regions which has a highly specific protease cleavage site and no low-specificity protease cleavage sites. For purposes of the present invention, a highly specific protease cleavage site has three or more specific adjacent amino acid residues that are recognized by the highly specific protease in order to permit cleavage (e.g., an enterokinase cleavage site). In contrast, a low-specificity protease cleavage site has two or less adjacent amino acid residues that are recognized by a protease in order to enable cleavage (e.g., a trypsin cleavage site).
In all seven BoNT serotypes, the amino acid preceding the N-terminus of the heavy chain is a Lys or Arg residue which is susceptible to proteolysis with trypsin. This trypsin-susceptible site can be replaced with a five amino acid enterokinase cleavage site (i.e., DDDDK (SEQ ID NO: 24)) upstream of the heavy chain's N-terminus, as illustrated for the seven serotypes in FIG. 2. This modification enables standardization activation with enterokinase. In serotypes A and C, additional Lys residues within this region are mutated to either Gln or His, thereby eliminating additional trypsin-susceptible sites which might result in undesirable non-specific activation of the toxin. Trypsin-susceptible recognition sequences also occur upstream of the heavy chain's receptor-binding domain in serotypes A, E, and F. This region's susceptibility to proteolysis is consistent with its exposure to solvent in the toxin's 3-D structure, as shown by X-ray crystallography analysis. Therefore, in serotypes A, E, and F, the susceptible residues are modified to Asn (FIG. 2). Signal peptides and N-terminal affinity tags are also preferably introduced, as required, to enable secretion and recovery.
In a preferred embodiment, the isolated Clostridial neurotoxin propeptide of the present invention has light and heavy chain regions which are not truncated.
As described in greater detail infra, the isolated Clostridial neurotoxin propeptide of the present invention may include a disabling mutation in an active metalloprotease site of the propeptide. The amino acid residues constituting the minimal catalytic domain of the light chain of the propeptide are illustrated in FIG. 1 and FIG. 2 by hatching. Specific amino acid residues constituting the active site of the catalytic domain of the metalloprotease are marked by stars in FIG. 1 and FIG. 2.
The Clostridial neurotoxin propeptide of the present invention may also possess a non-native motif in the light chain region that is capable of inactivating light chain metalloprotease activity in a toxic Clostridial neurotoxin. Suitable non-native motifs capable of inactivating light chain metalloprotease activity of a toxic Clostridial neurotoxin include, without limitation, SNARE motifs, metalloprotease inhibitor motifs, such as those present in the protein family known as Tissue Inhibitors of Metalloprotease (TIMP) (Mannello et al., “Matrix Metalloproteinase Inhibitors as Anticancer Therapeutics,” Curr. Cancer Drug Targets 5:285-298 (2005); Emonard et al., “Regulation of Matrix Metalloproteinase (MMP) Activity by the Low-Density Lipoprotein Receptor-Related Protein (LRP). A New Function for an ‘Old Friend,’” Biochimie 87:369-376 (2005); Maskos, “Crystal Structures of MMPs in Complex with Physiological and Pharmacological Inhibitors,” Biochimie 87:249-263 (2005), which are hereby incorporated by reference in their entirety), zinc chelating motifs based on suitably positioned sulfhydryl (preferably methionine) and acidic amino acids which become exposed upon binding of the chimeric antagonist to the active LC metalloprotease, and peptide motifs corresponding to the cleavage site on the substrate of LC metalloproteases, including transition state analogs of said cleavage site (Sukonpan et al., “Synthesis of Substrates and Inhibitors of Botulinum Neurotoxin Type A Metalloprotease,” J. Peptide Res. 63:181-193 (2004); Hayden et al., “Discovery and Design of Novel Inhibitors of Botulinus Neurotoxin A: Targeted ‘Hinge’ Peptide Libraries,” Journal of Applied Toxicology 23:1-7 (2003); Oost et al., “Design and Synthesis of Substrate-Based Inhibitors of Botulinum Neurotoxin Type B Metalloprotease,” Biopolymers (Peptide Science) 71:602-619 (2003), which are hereby incorporated by reference in its entirety).
SNARE motif peptides have been shown to prevent cleavage of synaptic complex components in Aplysia neurons (Rosetto et al., “SNARE Motif and Neurotoxins,” Nature 372:415-416 (1994), which is hereby incorporated by reference in its entirety). SNARE motif peptides are common to the substrate binding site of known BoNT serotypes, and have been shown to inhibit the toxic LC when injected into BoNT-affected neurons (Rosetto et al., “SNARE Motif and Neurotoxins,” Nature 372:415-416 (1994), which is hereby incorporated by reference in its entirety).
In a preferred embodiment, the Clostridial neurotoxin propeptide light chain region has one or more non-native motifs (e.g., SNARE motif peptides), which replace surface alpha-helix domains of the native propeptide. Seven surface alpha-helix domains in the light chain region of Clostridium botulinum serotypes are identified in FIG. 11.
A variety of Clostridial neurotoxin propeptides with light chain regions containing non-native motifs (e.g., SNARE motif peptides) in place of surface alpha-helix domains can be created. As described in greater detail below, these non-native motif bearing propeptides are generated by altering the nucleotide sequences of nucleic acids encoding the Clostridial neurotoxin propeptides.
Another aspect of the present invention relates to an isolated nucleic acid molecule encoding an isolated Clostridial neurotoxin propeptide of the present invention.
Nucleic acid molecules encoding full-length toxic Clostridial neurotoxins are well known in the art (See e.g., GenBank Accession Nos. M81186 (BoNT B); D90210 (BoNT C); S49407 (BoNT D); D90210 (BoNT E); X81714 (BoNT F); and X74162 (BoNT G)).
Nucleic acid molecules of the present invention preferably encode the amino acid sequences of FIG. 2. In particular, the nucleic acid molecules of the present invention are modified from the wild type BoNT serotype sequences to have one or more characteristics selected from the group consisting of a mutation which renders the encoded propeptide resistant to low-specificity proteolysis, one or more silent mutations that inactivate putative internal DNA regulatory elements, and one or more unique restriction sites. In particular, and as illustrated for each BoNT serotype in FIG. 2, mature neurotoxin stability and yield are optimized by site-directed mutation of residues within the intermediate region of the propeptide, thereby reducing the propeptides' susceptibility to non-specific proteolysis and poisoning of the host organism used for expression by the mature neurotoxin. Also, silent mutations are introduced into DNA regulatory elements that can affect RNA transcription or expression of the Clostridial neurotoxin propeptide in the system of choice. In addition, unique endonuclease restriction sites are introduced to enable creation of chimeric proteins.
A nucleic acid molecule of the present invention may also have a disabling mutation in a region encoding an active metalloprotease site of the propeptide, as described supra.
A nucleic acid molecule of the present invention may also have a mutation in a region encoding the light chain region, such that the nucleic acid molecule encodes, in the light chain region, a non-native motif capable of inactivating light chain metalloprotease activity in a toxic clostridial neurotoxin. Suitable non-native motifs are described supra.
A further aspect of the present invention relates to an expression system having a nucleic acid molecule encoding an isolated Clostridial neurotoxin propeptide of the present invention in a heterologous vector.
Yet another aspect of the present invention relates to a host cell having a heterologous nucleic acid molecule encoding an isolated Clostridial neurotoxin propeptide of the present invention.
Still another aspect of the present invention relates to a method of expressing a recombinant physiologically active Clostridial neurotoxin of the present invention. This method involves providing a nucleic acid construct having a nucleic acid molecule encoding an isolated Clostridial neurotoxin propeptide of the present invention. The nucleic acid construct has a heterologous promoter operably linked to the nucleic acid molecule and a 3′ regulatory region operably linked to the nucleic acid molecule. The nucleic acid construct is then introduced into a host cell under conditions effective to express the physiologically active Clostridial neurotoxin.
In a preferred embodiment, the expressed neurotoxin is contacted with a highly specific protease under conditions effective to effect cleavage at the intermediate region. Preferably, the intermediate region of the Clostridial neurotoxin propeptide is not cleaved by proteases endogenous to the expression system or the host cell.
Expression of a Clostridial neurotoxin of the present invention can be carried out by introducing a nucleic acid molecule encoding a Clostridial neurotoxin propeptide into an expression system of choice using conventional recombinant technology. Generally, this involves inserting the nucleic acid molecule into an expression system to which the molecule is heterologous (i.e., not normally present). The introduction of a particular foreign or native gene into a mammalian host is facilitated by first introducing the gene sequence into a suitable nucleic acid vector. “Vector” is used herein to mean any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which is capable of transferring gene sequences between cells. Thus, the term includes cloning and expression vectors, as well as viral vectors. The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′→3′) orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted Clostridial neurotoxin propeptide-coding sequences.
U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, including vaccinia virus, adenovirus, and retroviruses, including lentivirus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/− or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pFastBac series (Invitrogen), pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology Vol. 185 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety.
A variety of host-vector systems may be utilized to express the Clostridial neurotoxin propeptide-encoding sequence in a cell. Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation).
Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference in its entirety.
Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the PH promoter, T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5′ promoter elements may be used.
The Clostridial neurotoxin-encoding nucleic acid, a promoter molecule of choice, a suitable 3′ regulatory region, and if desired, a reporter gene, are incorporated into a vector-expression system of choice to prepare a nucleic acid construct using standard cloning procedures known in the art, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York (2001), which is hereby incorporated by reference in its entirety.
The nucleic acid molecule encoding a Clostridial neurotoxin is inserted into a vector in the sense (i.e., 5′→3′) direction, such that the open reading frame is properly oriented for the expression of the encoded Clostridial neurotoxin propeptide under the control of a promoter of choice. Single or multiple nucleic acids may be ligated into an appropriate vector in this way, under the control of a suitable promoter, to prepare a nucleic acid construct.
Once the isolated nucleic acid molecule encoding the Clostridial neurotoxin propeptide has been inserted into an expression vector, it is ready to be incorporated into a host cell. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, lipofection, protoplast fusion, mobilization, particle bombardment, or electroporation. The DNA sequences are incorporated into the host cell using standard cloning procedures known in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety. Suitable hosts include, but are not limited to, bacteria, virus, yeast, fungi, mammalian cells, insect cells, plant cells, and the like. Preferable host cells of the present invention include, but are not limited to, Escherichia coli, insect cells, and Pichia pastoris cells.
Typically, an antibiotic or other compound useful for selective growth of the transformed cells only is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present in the plasmid with which the host cell was transformed. Suitable genes are those which confer resistance to gentamycin, G418, hygromycin, puromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like. Similarly, “reporter genes” which encode enzymes providing for production of an identifiable compound, or other markers which indicate relevant information regarding the outcome of gene delivery, are suitable. For example, various luminescent or phosphorescent reporter genes are also appropriate, such that the presence of the heterologous gene may be ascertained visually.
In a preferred embodiment of the present invention, the expressed neurotoxin propeptide is contacted with a highly specific protease (e.g., enterokinase) under conditions effective to enable cleavage at the intermediate region of the propeptide of the present invention. Preferably, the expressed neurotoxin propeptide has one or more disulfide bridges.
Another aspect of the present invention relates to an isolated, physiologically active Clostridial neurotoxin produced by cleaving an isolated Clostridial neurotoxin propeptide of the present invention. The propeptide is cleaved at the highly specific protease cleavage site. The light and heavy chain regions are linked by a disulfide bond.
As discussed supra, Clostridial neurotoxins are synthesized as single chain propeptides which are later activated by a specific proteolysis cleavage event, generating a dimer joined by a disulfide bond. These structural features can be illustrated using BoNT A as an example, and are generally applicable to all Clostridium botulinum serotypes. The mature BoNT A is composed of three functional domains of Mr ̃50,000 (FIG. 3A), where the catalytic function responsible for toxicity is confined to the light chain (residues 1-437), the translocation activity is associated with the N-terminal half of the heavy chain (residues 448-872), and cell binding is associated with its C-terminal half (residues 873-1,295) (Johnson, “Clostridial Toxins as Therapeutic Agents: Benefits of Nature's Most Toxic Proteins,” Annu. Rev. Microbiol. 53:551-575 (1999); Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995), which are hereby incorporated by reference in their entirety).
Optimized expression and recovery of recombinant neurotoxins for BoNT serotypes in a native and physiologically active state is achieved by the introduction of one or more alterations to the nucleotide sequences encoding the BoNT propeptides, as discussed supra. These mutations are designed to maximize yield of recombinant Clostridial neurotoxin, while retaining the native toxins structure and biological activity.
Isolated, full-length Clostridial neurotoxins of the present invention are physiologically active. This physiological activity includes, but is not limited to, toxin immunogenicity, trans- and intra-cellular trafficking, and cell recognition.
The mechanism of cellular binding and internalization of Clostridial toxins is still poorly understood. No specific receptor has been unambiguously identified, and the binding constants have not been characterized. The C-terminal portion of the heavy chain of all Clostridial neurotoxins binds to gangliosides (sialic acid-containing glycolipids), with a preference for gangliosides of the G1b series (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Montecucco, “How Do Tetanus and Botulinum Toxins Bind to Neuronal Membranes?” TIBS 11:314-317 (1986); and Van Heyningen et al., “The Fixation of Tetanus Toxin by Ganglioside,” J. Gen. Microbiol. 24:107-119 (1961), which are hereby incorporated by reference in their entirety). The sequence responsible for ganglioside binding has been identified for the structurally similar TeNT molecule, and is located within the 34 C-terminal amino acid residues of its heavy chain. BoNT A, B, C, E, and F share a high degree of homology with TeNT in this region (FIG. 1) (Shapiro et al., “Identification of a Ganglioside Recognition Domain of Tetanus Toxin Using a Novel Ganglioside Photoaffinity Ligand,” J. Biol. Chem. 272:30380-30386 (1997), which is hereby incorporated by reference in its entirety). Multiple types of evidence suggest the existence of at least one additional component involved in the binding of Clostridial neurotoxins to neuronal membranes (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Montecucco, “How Do Tetanus and Botulinum Toxins Bind to Neuronal Membranes?” TIBS 11:314-317 (1986), which are hereby incorporated by reference in their entirety). In two reports (Nishiki et al., “The High-Affinity Binding of Clostridium Botulinum Type B Neurotoxin to Synaptotagmin II Associated with Gangliosides GT1b/GD1a,” FEBS Lett. 378:253-257 (1996); Dong et al., “Synaptotagmins I and II Mediate Entry of Botulinum Neurotoxin B into Cells,” J. Cell Biol. 162:1293-1303 (2003), which are hereby incorporated by reference in their entirety), synaptotagmins were identified as possible candidates for the auxiliary BoNT B receptor, and synaptotagmins I and II were implicated as neuronal receptors for BoNT G (Rummel et al., “Synaptotagmins I and II Act as Nerve Cell Receptors for Botulinum Neurotoxin G,” J. Biol. Chem. 279:30865-30870 (2004), which is hereby incorporated by reference in its entirety). However despite the structural similarity in the putative receptor-binding domain of Clostridial neurotoxins, other toxin subtypes show no affinity for synaptotagmins or synaptotagmin-related molecules. Lipid rafts (Herreros et al., “Lipid Rafts Act as Specialized Domains for Tetanus Toxin Binding and Internalization into Neurons,” Mol. Biol. Cell 12:2947-2960 (2001), which is hereby incorporated by reference in its entirety) have been implicated as a specialized domain involved in TeNT binding and internalization into neurons, but these domains are widely distributed on multiple cell types, and therefore cannot simply explain the high specificity of the toxins for neurons.
Clostridial neurotoxins are internalized through the presynaptic membrane by an energy-dependent mechanism (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Matteoli et al., “Synaptic Vesicle Endocytosis Mediates the Entry of Tetanus Neurotoxin into Hippocampal Neurons,” Proc. Natl. Acad. Sci. USA 93:13310-13315 (1996); and Mukherjee et al., “Endocytosis,” Physiol. Rev. 77:759-803 (1997), which are hereby incorporated by reference in their entirety), and rapidly appear in vesicles where they are at least partially protected from degradation (Dolly et al., “Acceptors for Botulinum Neurotoxin Reside on Motor Nerve Terminals and Mediate Its Internalization,” Nature 307:457-460 (1984); Critchley et al., “Fate of Tetanus Toxin Bound to the Surface of Primary Neurons in Culture: Evidence for Rapid Internalization,” J. Cell Biol. 100:1499-1507 (1985), which are hereby incorporated by reference in their entirety). The BoNT complex of light and heavy chains interacts with the endocytic vesicle membrane in a chaperone-like way, preventing aggregation and facilitating translocation of the light chain in a fashion similar to the protein conducting/translocating channels of smooth ER, mitochondria, and chloroplasts (Koriazova et al., “Translocation of Botulinum Neurotoxin Light Chain Protease through the Heavy Chain Channel,” Nat. Struct. Biol. 10:13-18 (2003), which is hereby incorporated by reference in its entirety). Acidification of the endosome is believed to induce pore formation, which allows translocation of the light chain to the cytosol upon reduction of the interchain disulfide bond (Hoch et al., “Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins Across Membranes,” Proc. Natl. Acad. Sci. USA 82:1692-1696 (1985), which is hereby incorporated by reference in its entirety). Within the cytosol, the light chain displays a zinc-endopeptidase activity specific for protein components of the synaptic vesicle exocytosis apparatus. TeNT and BoNT B, D, F, and G recognize VAMP/synaptobrevin. This integral protein of the synaptic vesicle membrane is cleaved at a single peptide bond, which differs for each neurotoxin. BoNT A, C, and E recognize and cleave SNAP-25, a protein of the presynaptic membrane, at two different sites within the carboxyl terminus. BoNT C also cleaves syntaxin, another protein of the nerve plasmalemma (Montecucco et al., “Structure and Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Sutton et al., “Crystal Structure of a SNARE Complex Involved in Synaptic Exocytosis at 2.4 {acute over (Å)} Resolution,” Nature 395:347-353 (1998), which are hereby incorporated by reference in their entirety). The cleavage of any component of the synaptic release machinery results in inhibition of acetylcholine release, ultimately leading to neuromuscular paralysis.
In one embodiment of the present invention, the isolated Clostridial neurotoxin is toxic. The toxicity of Clostridial neurotoxins is a result of a multi-step mechanism. From the circulation, BoNT targets the pre-synaptic membrane of neuromuscular junctions, where it is internalized to directly exert its toxic effect on the peripheral nervous system (Dolly et al., “Acceptors for Botulinum Neurotoxin Reside on Motor Nerve Terminals and Mediate Its Internalization,” Nature 307:457-460 (1984), which is hereby incorporated by reference in its entirety). Toxicity at the neuromuscular junction involves neuron binding; internalization into endocytic vesicles, similar to those involved in synaptic vesicle recycling; activation within an acidic compartment to the proteolytically active toxin which then penetrates into the neuronal cytoplasm; and target recognition and catalytic cleavage of substrates in the neuronal machinery for synaptic vesicle exocytosis.
In an alternative embodiment of the present invention, the isolated Clostridial neurotoxin is physiologically active and atoxic. The endopeptidase activity responsible for Clostridial neurotoxin toxicity is believed to be associated with the presence of a HExxHxxH (SEQ ID NO: 25) motif in the light chain, characteristic of metalloproteases (FIG. 1). Mutagenesis of BoNT A light chain, followed by microinjection of the corresponding mRNA into presynaptic cholinergic neurons of Aplysia californica, allowed the minimal essential domain responsible for toxicity to be identified (Kurazono et al., “Minimal Essential Domains Specifying Toxicity of the Light Chains of Tetanus Toxin and Botulinum Neurotoxin Type A,” J. Biol. Chem. 267:14721-14729 (1992), which is hereby incorporated by reference in its entirety). Site-directed mutagenesis of BoNT A light chain pinpointed the amino acid residues involved in Zn2 coordination, and formation of the active metalloendoprotease core which cleaves SNAP-25 (Rigoni et al., “Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun. 288:1231-1237 (2001), which is hereby incorporated by reference in its entirety). The three-dimensional structures of Clostridial neurotoxins and their derivatives confirmed the mutagenesis results, and detailed the spatial organization of the protein domains. For the BoNT A holotoxin, crystal structure was obtained to a resolution of 3.3 {acute over (Å)} (Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998), which is hereby incorporated by reference in its entirety). The BoNT B holotoxin crystal structure was determined at 1.8 and 2.6 Å resolution (Swaminathan et al., “Structural Analysis of the Catalytic and Binding Sites of Clostridium Botulinum Neurotoxin B,” Nat. Struct. Biol. 7:693-699 (2000), which is hereby incorporated by reference in its entirety). Recently, a crystal structure for BoNT E catalytic domain was determined to 2.1 {acute over (Å)} resolution (Agarwal et al., “Structural Analysis of Botulinum Neurotoxin Type E Catalytic Domain and Its Mutant Glu212>Gln Reveals the Pivotal Role of the Glu212 Carboxylate in the Catalytic Pathway,” Biochemistry 43:6637-6644 (2004), which is hereby incorporated by reference in its entirety). The later study provided multiple interesting structural details, and helps explain the complete loss of metalloendoproteolytic activity in the BoNT E LC E212>Q mutant. The availability of this detailed information on the relationship between the amino acid sequence and biological activities of Clostridial toxins enables the design of modified toxin genes with properties specifically altered for therapeutic goals.
Thus, in a preferred embodiment, the physiologically active and atoxic Clostridial neurotoxin of the present invention has a disabling mutation in an active metalloprotease site.
The physiologically active and atoxic Clostridial neurotoxin of the present invention may also have a non-native motif (e.g., a SNARE motif) in the light chain region that is capable of inactivating light chain metalloprotease activity in a toxic Clostridial neurotoxin. FIG. 11 illustrates the sequences of nine chimeric proteins, which are physiologically active and atoxic Clostridial neurotoxins containing at least one non-native motif in the light chain region that is capable of inactivating light chain metalloprotease activity in a toxic Clostridial neurotoxin. The non-native motifs are substituted for alpha-helix domains. When present in the physiologically active and atoxic Clostridial neurotoxin, the non-native protein motifs enable the neurotoxin to bind, inactivate, or otherwise mark the toxic light chain region of a wild type Clostridial neurotoxin for elimination from the cytosol of neurotoxin-affected neurons. As such, a physiologically active and atoxic Clostridial neurotoxin having a non-native motif in the light chain region that is capable of inactiving light chain metalloprotease activity in a toxic Clostridial neurotoxin is useful as an antidote to effectively target the cytoplasm of neurotoxin-affected neurons. Administration of such antidotes is described in greater detail below.
Yet a further aspect of the present invention relates to a vaccine or antidote having an isolated, physiologically active, atoxic, Clostridial neurotoxin produced by cleaving an isolated Clostridial neurotoxin propeptide of the present invention. The propeptide is cleaved at the highly specific protease cleavage site. The light and heavy chain regions are linked by a disulfide bond.
Developing effective vaccines and antidotes against Clostridial neurotoxins requires the preservation of structural features important to toxin trafficking and immunogenicity. From a practical perspective, this is most easily achieved by first producing recombinant molecules that retain the structural features and toxicity of native toxin, followed by selective modification to eliminate toxicity and introduce therapeutic utility. To achieve this goal, a versatile platform for the genetic manipulation of Clostridial toxin genes and for their selective modification was developed (described infra). The genetic engineering scheme can produce full-length toxic and atoxic derivatives of BoNT A, which retains important aspects of the wild toxin's native structure. This methodology can be generalized across the entire family of Clostridial neurotoxins because of their structural similarities (See FIGS. 1-2).
Thus, in a preferred embodiment, the vaccine or antidote of the present invention is a physiologically active and atoxic Clostridial neurotoxin from Clostridium botulinum, such as from Clostridium botulinum serotypes A-G. As described supra, the vaccine or antidote has the physiological activity of a wild Clostridial neurotoxin, which activity includes, but is not limitated to, toxin immunogenicity, trans- and intra-cellular trafficking, and cell recognition. The Clostridial neurotoxin of the vaccine or antidote is rendered atoxic by a mutation in its active metalloprotease site, as described supra. Additional mutuations may be introduced to ensure atoxicity and introduce new biological activities, while preserving systemic trafficking and cellular targeting of the vaccine or antidote. As has also been described, the vaccine or antidote may possess non-native motifs in the light chain region that are capable of inactivating light chain metalloprotease activity in a toxic Clostridial neurotoxin.
Atoxic Clostridial neurotoxins can be tested as candidate vaccines and antidotes to BoNT poisoning. Atoxic derivatives are created using the BoNT toxic derivative constructs developed under the methods described infra. Point mutations are introduced into the toxin's active metalloprotease site to eliminate toxicity while maintaining native toxin structure, immunogenicity, trans- and intra-cellular trafficking, and cell recognition. Expression systems and purification schemes are optimized as described infra. Derivatives found to completely lack toxicity yet retain relevant biological activities of the native toxin, are evaluated for their potential as either vaccines or antidotes to BoNT poisoning. Parenteral routes of administration are tested first, followed by evaluation of oral and inhalational routes as applicable. Utility as a vaccine is determined by immunogenicity and challenge studies in mice. Utility as an antidote is first evaluated in vitro by testing the ability of atoxic derivatives to prevent neuromuscular blockade in the mouse phrenic-nerve hemidiaphragm, and to inhibit native toxin trafficking in the transcytosis assay. Effective in vitro antagonists are tested as in vivo antidotes, and may be superior to antibody-based antidotes because they more effectively mimic native toxin absorption and trafficking pathways. Antidote effectiveness in vivo is first evaluated using simultaneous dosing. Additional dosage and timing parameters relevant to using antidotes under crisis situations is further evaluated for atoxic derivatives found to be effective when administered simultaneously with toxin. Using these procedures, a series of atoxic derivatives and fusion proteins are created and their biological activities systematically catalogued. The availability of these well characterized BoNT gene constructs and toxin derivatives enables the rational design of new anti-BoNT therapeutics. Dose-response analyses and challenge studies against active neurotoxin provide data that allows the best candidate vaccines and antidotes to be selected for further development.
A further aspect of the present invention relates to method of immunizing a subject against toxic effects of a Clostridial neurotoxin. This method involves administering a vaccine of the present invention to the subject under conditions effective to immunize the subject against toxic effects of Clostridial neurotoxin.
The subject administered the vaccine may further be administered a booster of the vaccine under conditions effective to enhance immunization of the subject.
Another aspect of the present invention relates to a method of treating a subject for toxic effects of a Clostridial neurotoxin. This method involves administering an antidote comprising an isolated, physiologically active, atoxic, Clostridial neurotoxin produced by cleaving the isolated Clostridial neurotoxin propeptide of the present invention to the subject under conditions effective to treat the subject for toxic effects of Clostridial neurotoxin.
A vaccine or antidote of the present invention can be administered to a subject orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. The vaccine or antidote may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
The vaccine or antidote of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or may be enclosed in hard or soft shell capsules, or may be compressed into tablets, or may be incorporated directly with the food of the diet. For oral therapeutic administration, the vaccine or antidote may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compound in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 1 and 250 mg of active compound.
The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a fatty oil.
Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
The vaccine or antidote may also be administered parenterally. Solutions or suspensions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
The vaccine or antidote of the present invention may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the vaccine or antidote of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The vaccine or antidote of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
A further aspect of the present invention relates to a chimeric protein having a first protein or protein fragment having a heavy chain region of a Clostridial neurotoxin and a second protein or protein fragment linked to the first protein or protein fragment.
In a preferred embodiment, the second protein or protein fragment has therapeutic functionality which can target specific steps in a trafficking pathway of the Clostridial neurotoxin.
BoNTs pass across epithelial surfaces without being destroyed or causing local toxicity. Passage across epithelia is believed to occur by specific binding and transcytosis. The ability of intact BoNT A to pass though pulmonary epithelia and resist proteolytic inactivation was demonstrated in rat primary alveolar epithelial cells and in immortalized human pulmonary adenocarcinoma (Calu-3) cells. The rate of transport was greater in the apical-to-basolateral direction than in the basolateral-to-apical direction, and it was blocked by serotype-specific toxin antibodies (Park et al., “Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component,” Infect. Immun. 71:1147-1154 (2003), which is hereby incorporated by reference in its entirety).
The ability of Clostridial neurotoxins to pass undegraded through epithelial barriers via transcytosis and to specifically target nervous tissue makes Clostridial neurotoxins useful in the development of oral and inhalational carriers for therapeutic agents that cannot normally be delivered via these routes of administration, and as delivery vehicles which can specifically target the peripheral and central nervous system.
Still another aspect of the present invention relates to a treatment method. This method involves contacting a patient with an isolated, physiologically active, toxic, Clostridial neurotoxin produced by cleaving an isolated Clostridial neurotoxin propeptide according to the present invention, under conditions effective to treat the patient.
By treatment, it is meant aesthetic treatment (See e.g., Carruthers et al., “Botulinum Toxin A in the Mid and Lower Face and Neck,” Dermatol. Clin. 22:151-158 (2004); Lang, “History and Uses of BOTOX (Botulinum Toxin Type A),” Lippincotts Case Manag. 9:109-112 (2004); Naumann et al., “Safety of Botulinum Toxin Type A: A Systematic Review and Meta-Analysis,” Curr. Med. Res. Opin. 20:981-990 (2004); Vartanian et al., “Facial Rejuvenation Using Botulinum Toxin A: Review and Updates,” Facial Plast. Surg. 20:11-19 (2004), which are hereby incorporated by reference in their entirety) as well as therapeutic treatment (See e.g., Bentsianov et al., “Noncosmetic Uses of Botulinum Toxin,” Clin. Dermatol. 22:82-88 (2004); Carruthers et al., “Botox: Beyond Wrinkles,” Clin. Dermatol. 22:89-93 (2004); Jankovic, “Botulinum Toxin In Clinical Practice,” J. Neurol. Neurosurg. Psychiatry 75:951-957 (2004); Klein, “The Therapeutic Potential of Botulinum Toxin,” Dermatol. Surg. 30:452-455 (2004); Schurch, “The Role of Botulinum Toxin in Neurology,” Drugs Today (Barc) 40:205-212 (2004), which are hereby incorporated by reference in their entirety).
Preferred treatment methods of the present invention include, but are not limited to, dermatologic, gastroenterologic, genitourinaric, and neurologic treatment.
Dermatologic treatment includes, but is not limited to, treatment for Rhtyiddess (wrinkles) (Sadick et al., “Comparison of Botulinum Toxins A and B in the Treatment of Facial Rhytides,” Dermatol. Clin. 22:221-226 (2004), which is hereby incorporated by reference in its entirety), including glabellar (Carruthers et al., “Botulinum Toxin type A for the Treatment of Glabellar Rhytides,” Dermatol. Clin. 22:137-144 (2004); Ozsoy et al., “Two-Plane Injection of Botulinum Exotoxin A in Glabellar Frown Lines,” Aesthetic Plast. Surg. 28:114-115 (2004); which are hereby incorporated by reference in their entirety), neck lines (Brandt et al., “Botulinum Toxin for the Treatment of Neck Lines and Neck Bands,” Dermatol. Clin. 22:159-166 (2004), which is hereby incorporated by reference in its entirety), crows feet (Levy et al., “Botulinum Toxin A: A 9-Month Clinical and 3D In Vivo Profilometric Crow's Feet Wrinkle Formation Study,” J. Cosmet. Laser Ther. 6:16-20 (2004), which is hereby incorporated by reference in its entirety), and brow contour (Chen et al., “Altering Brow Contour with Botulinum Toxin,” Facial Plast. Surg. Clin. North Am. 11:457-464 (2003), which is hereby incorporated by reference in its entirety). Other dermatologic treatment includes treatment for hypertrophic masateer muscles in Asians (Ahn et al., “Botulinum Toxin for Masseter Reduction in Asian Patients,” Arch. Facial Plast. Surg. 6:188-191 (2004), which is hereby incorporated by reference in its entirety) and focal hyperhydrosis (Glogau, “Treatment of Hyperhidrosis with Botulinum Toxin,” Dermatol. Clin. 22:177-185, vii (2004), which is hereby incorporated by reference in its entirety), including axillary (“Botulinum Toxin (Botox) for Axillary Hyperhidrosis,” Med. Lett. Drugs Ther. 46:76 (2004), which is hereby incorporated by reference in its entirety) and genital (Lee et al., “A Case of Foul Genital Odor Treated with Botulinum Toxin A,” Dermatol. Surg. 30:1233-1235 (2004), which is hereby incorporated by reference in its entirety).
Gastroentologic treatment includes, but is not limited to, treatment for esophageal motility disorders (Achem, “Treatment of Spastic Esophageal Motility Disorders,” Gastroenterol. Clin. North Am. 33:107-124 (2004), which is hereby incorporated by reference in its entirety), pharyngeal-esophageal spasm (Bayles et al., “Operative Prevention and Management of Voice-Limiting Pharyngoesophageal Spasm,” Otolaryngol. Clin. North Am. 37:547-558 (2004); Chao et al., “Management of Pharyngoesophageal Spasm with Botox,” Otolaryngol. Clin. North Am. 37:559-566 (2004), which are hereby incorporated by reference in their entirety), and anal fissure (Brisinda et al., “Botulinum Neurotoxin to Treat Chronic Anal Fissure: Results of a Randomized ‘Botox vs. Dysport’ Controlled Trial,” Ailment Pharmacol. Ther. 19:695-701 (2004); Jost et al., “Botulinum Toxin A in Anal Fissure: Why Does it Work?” Dis. Colon Rectum 47:257-258 (2004), which are hereby incorporated by reference in their entirety).
Genitourinaric treatment includes, but is not limited to, treatment for neurogenic dysfunction of the urinary tract (“Botulinic Toxin in Patients with Neurogenic Dysfunction of the Lower Urinary Tracts,” Urologia July-August:44-48 (2004); Giannantoni et al., “Intravesical Resiniferatoxin Versus Botulinum-A Toxin Injections for Neurogenic Detrusor Overactivity: A Prospective Randomized Study,” J. Urol. 172:240-243 (2004); Reitz et al., “Intravesical Therapy Options for Neurogenic Detrusor Overactivity,” Spinal Cord 42:267-272 (2004), which are hereby incorporated by reference in their entirety), overactive bladder (Cruz, “Mechanisms Involved in New Therapies for Overactive Bladder,” Urology 63:65-73 (2004), which is hereby incorporated by reference in its entirety), and neuromodulation of urinary urge incontinence (Abrams, “The Role of Neuromodulation in the Management of Urinary Urge Incontinence,” BJU Int. 93:1116 (2004), which is hereby incorporated by reference in its entirety).
Neurologic treatment includes, but is not limited to, treatment for tourettes syndrome (Porta et al., “Treatment of Phonic Tics in Patients with Tourette's Syndrome Using Botulinum Toxin Type A,” Neurol. Sci. 24:420-423 (2004), which is hereby incorporated by reference in its entirety) and focal muscle spasticity or dystonias (MacKinnon et al., “Corticospinal Excitability Accompanying Ballistic Wrist Movements in Primary Dystonia,” Mov. Disord. 19:273-284 (2004), which is hereby incorporated by reference in its entirety), including, but not limited to, treatment for cervical dystonia (Haussermann et al., “Long-Term Follow-Up of Cervical Dystonia Patients Treated with Botulinum Toxin A,” Mov. Disord. 19:303-308 (2004), which is hereby incorporated by reference in its entirety), primary blepharospasm (Defazio et al., “Primary Blepharospasm: Diagnosis and Management,” Drugs 64:237-244 (2004), which is hereby incorporated by reference in its entirety), hemifacial spasm, post-stroke (Bakheit, “Optimising the Methods of Evaluation of the Effectiveness of Botulinum Toxin Treatment of Post-Stroke Muscle Spasticity,” J. Neurol. Neurosurg. Psychiatry 75:665-666 (2004), which is hereby incorporated by reference in its entirety), spasmodic dysphonia (Bender et al., “Speech Intelligibility in Severe Adductor Spasmodic Dysphonia,” J. Speech Lang. Hear Res. 47:21-32 (2004), which is hereby incorporated by reference in its entirety), facial nerve disorders (Finn, “Botulinum Toxin Type A: Fine-Tuning Treatment of Facial Nerve Injury,” J. Drugs Dermatol. 3:133-137 (2004), which is hereby incorporated by reference in its entirety), and Rasmussen syndrome (Lozsadi et al., “Botulinum Toxin A Improves Involuntary Limb Movements in Rasmussen Syndrome,” Neurology 62:1233-1234 (2004), which is hereby incorporated by reference in its entirety). Other neurologic treatments include treatment for amputation pain (Kern et al., “Effects of Botulinum Toxin Type B on Stump Pain and Involuntary Movements of the Stump,” Am. J. Phys. Med. Rehabil. 83:396-399 (2004), which is hereby incorporated by reference in its entirety), voice tremor (Adler et al., “Botulinum Toxin Type A for Treating Voice Tremor,” Arch. Neurol. 61:1416-1420 (2004), which is hereby incorporated by reference in its entirety), crocodile tear syndrome (Kyrmizakis et al., “The Use of Botulinum Toxin Type A in the Treatment of Frey and Crocodile Tears Syndrome,” J. Oral Maxillofac. Surg. 62:840-844 (2004), which is hereby incorporated by reference in its entirety), marginal mandibular nerve paralysis, and pain control (Cui et al., “Subcutaneous Administration of Botulinum Toxin A Reduces Formalin-Induced Pain,” Pain 107:125-133 (2004), which is hereby incorporated by reference in its entirety), including but not limited to pain after mastectomy (Layeeque et al., “Botulinum Toxin Infiltration for Pain Control After Mastectomy and Expander Reconstruction,” Ann. Surg. 240:608-613 (2004), which is hereby incorporated by reference in its entirety) and chest pain of esophageal origin (Schumulson et al., “Current and Future Treatment of Chest Pain of Presumed Esophageal Origin,” Gastroenterol. Clin. North Am. 33:93-105 (2004), which is hereby incorporated by reference in its entirety). Another neurologic treatment amenable to the methods of the present invention is headache (Blumenfeld et al., “Botulinum Neurotoxin for the Treatment of Migraine and Other Primary Headache Disorders,” Dermatol. Clin. 22:167-175 (2004), which is hereby incorporated by reference in its entirety).
The methods of the present invention are also suitable for treatment of cerebral palsy (Balkrishnan et al., “Longitudinal Examination of Health Outcomes Associated with Botulinum Toxin Use in Children with Cerebral Palsy,” J. Surg. Orthop. Adv. 13:76-80 (2004); Berweck et al., “Use of Botulinum Toxin in Pediatric Spasticity (Cerebral Palsy),” Mov. Disord. 19:S162-S167 (2004); Pidcock, “The Emerging Role of Therapeutic Botulinum Toxin in the Treatment of Cerebral Palsy,” J. Pediatr. 145:S33-S35 (2004), which are hereby incorporated by reference in their entirety), hip adductor muscle dysfunction in multiple sclerosis (Wissel et al., “Botulinum Toxin Treatment of Hip Adductor Spasticity in Multiple Sclerosis,” Wien Klin Wochesnchr 4:20-24 (2001), which is hereby incorporated by reference in its entirety), neurogenic pain and inflammation, including arthritis, iatrogenic parotid sialocele (Capaccio et al., “Diagnosis and Therapeutic Management of Iatrogenic Parotid Sialocele,” Ann. Otol. Rhinol. Laryngol. 113:562-564 (2004), which is hereby incorporated by reference in its entirety), and chronic TMJ displacement (Aquilina et al., “Reduction of a Chronic Bilateral Temporomandibular Joint Dislocation with Intermaxillary Fixation and Botulinum Toxin A,” Br. J. Oral Maxillofac. Surg. 42:272-273 (2004), which is hereby incorporated by reference in its entirety). Other conditions that can be treated by local controlled delivery of pharmaceutically active toxin include intra-articular administration for the treatment of arthritic conditions (Mahowald et al., “Long Term Effects of Intra-Articular BoNT A for Refractory Joint Pain,” Annual Meeting of the American College of Rheumatology (2004), which is hereby incorporated by reference in its entirety), and local administration for the treatment of joint contracture (Russman et al., “Cerebral Palsy: A Rational Approach to a Treatment Protocol, and the Role of Botulinum Toxin in Treatment,” Muscle Nerve Suppl. 6:S181-S193 (1997); Pucinelli et al., “Botulinic Toxin for the Rehabilitation of Osteoarthritis Fixed-Flexion Knee Deformity,” Annual Meeting of the Osteoarthitis Research Society International (2004), which are hereby incorporated by reference in their entirety). The methods of the present invention are also suitable for the treatment of pain associated with various conditions characterized by the sensitization of nociceptors and their associated clinical syndromes, as described in Bach-Rojecky et al., “Antinociceptive Effect of Botulinum Toxin Type A In Rat Model of Carrageenan and Capsaicin Induced Pain,” Croat. Med. J. 46:201-208 (2005); Aoki, “Evidence for Antinociceptive Activity of Botulinum Toxin Type A in Pain Management,” Headache 43 Suppl 1:S9-15 (2003); Kramer et al., “Botulinum Toxin A Reduces Neurogenic Flare But Has Almost No Effect on Pain and Hyperalgesia in Human Skin,” J. Neurol. 250:188-193 (2003); Blersch et al., “Botulinum Toxin A and the Cutaneous Nociception in Humans: A Prospective, Double-Blind, Placebo-Controlled, Randomized Study,” J. Neurol. Sci. 205:59-63 (2002), which are hereby incorporated by reference in its entirety.
The methods and products of the present invention may be customized to optimize therapeutic properties (See e.g., Chaddock et al., “Retargeted Clostridial Endopeptidases Inhibition of Nociceptive Neurotransmitter Release In Vitro, and Antinociceptive Activity in In Vivo Models of Pain,” Mov. Disord. 8:S42-S47 (2004); Finn, “Botulinum Toxin Type A: Fine-Tuning Treatment of Facial Nerve Injury,” J. Drugs Dermatol. 3:133-137 (2004); Eleopra et al., “Different Types of Botulinum Toxin in Humans,” Mov. Disord. 8:S53-S59 (2004); Flynn, “Myobloc,” Dermatol. Clin. 22:207-211 (2004); and Sampaio et al., “Clinical Comparability of Marketed Formulations of Botulinum Toxin,” Mov. Disord. 8:S129-S136 (2004), which are hereby incorporated by reference in their entirety).
EXAMPLES
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Example 1
SDS PAGE
Samples from all intermediate purification steps, as well as pure recombinant protein, were routinely separated and visualized on 8% separating polyacrylamide gels, according to Laemmli procedure (Laemmli, “Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4,” Nature 227:680-685 (1970), which is hereby incorporated by reference in its entirety). Protein bands were visualized by Bio-Safe Coomassie G-250 Stain (Bio-Rad, Cat. #161-0786).
Example 2
Western Blotting
Samples for Western blot analysis were separated on 8% SDS-polyacrylamide gels. Followed by separation, proteins were transferred to the Hybond-C nitrocellulose membrane (Amersham Biosciences, Cat. #RPN303C) in 1× Tris/Glycine buffer (Bio-Rad, Cat. #161-0734) supplemented with 20% methanol at 100 volts for 2 hours, 4° C. After the transfer, membrane was rinsed in distilled water and protein bands were visualized by staining with 0.2% Ponceau S in 1% acetic acid for 1 minute. Dye from the membrane was washed away in the Tris-buffered saline/0.1% Tween-20 buffer, pH 7.5, followed by incubation of the membrane in the blocking reagent (5% non-fat powdered milk in Tris-buffered saline/0.1% Tween-20 buffer, pH 7.5) for 16 hours at 4° C. For immunodetection, membrane was incubated with primary antibodies/immune serum at 1:7,000 dilution, in 0.5% non-fat milk in Tris-buffered saline/0.1% Tween-20 buffer, pH 7.5 at room temperature for 2 hours. Membrane was washed (6×5 min) and incubated with secondary antibody at 1:10,000 dilution at room temperature for 25 minutes. After the series of additional washing (6×5 min), immunoreactive bands were visualized using ECL (enhanced chemiluminescence) Plus Western Blotting Reagent (Amersham Biosciences, Cat. #RPN2124) according to manufacturer instructions. Hyperfilm ECL (Amersham Biosciences, Cat. #RPN1674K) was used for autoradiography with the exposure time adequate to visualize chemiluminescent bands. The proteins were identified by comparison with the positive controls and molecular weight protein standards.
Example 3
Evaluation of Recombinant Toxin Yield
The protein concentration of the purified recombinant protein fractions were determined using the BCA Protein assay reagent (Pierce, Cat. #23225) with bovine serum albumin used as standard.
Example 4
In Vitro Toxicity Assay on the Mouse Phrenic Nerve-Hemidiaphragm Preparation
The toxicity of the various recombinant proteins is bioassayed on the mouse phrenic nerve-hemidiaphragm preparation (Simpson et al., “Isolation and Characterization of a Novel Human Monoclonal Antibody that Neutralizes Tetanus Toxin,” J. Pharmacol. Exp. Ther. 254:98-103 (1990), which is hereby incorporated by reference in its entirety). Tissues are excised and suspended in physiological buffer, aerated with 95% O2, 5% CO2, and maintained at 35° C. The physiological solution has the following composition: 137 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgSO4, 24 mM NaHCO3, 1 mM NaH2PO4, 11 mM D-glucose, and 0.01% gelatin. Phrenic nerves are stimulated continuously (1.0 Hz; 0.1-0.3 msec duration), and muscle twitch is recorded. Toxin-induced paralysis is measured as a 50% reduction in muscle twitch response to neurogenic stimulation.
Example 5
In Vitro Transcytosis Assay
Cells are grown on polycarbonate membranes with a 0.4 μm pore size in Transwell® porous bottom inserts (Corning-Costar) (FIG. 10) (Zweibaum et al., “Use of Cultured Cell Lines in Studies of Intestinal Cell Differentiation and Function,” In: Handbook of Physiology, Section 6: “The Gastrointestinal System,” Edited by Schulz et al., American Physiological Society, Bethesda, Vol. IV, 223-255; Dharmsathaphorn et al., “A Human Colonic Tumor Cell Line that Maintains Vectorial Electrolyte Transport,” Am. J. Physiol. 246:G204-G208 (1984); and Dharmsathaphorn et al., “Established Intestinal Cell Lines as Model Systems for Electrolyte Transport Studies,” Methods Enzymol. 192:354-389 (1990), which are hereby incorporated by reference in their entirety). The cell growth area within each insert is equivalent to 1 cm2. Prior to seeding the cells, insert membranes are coated with 10 μg/cm2 rat tail collagen type I. Collagen stock solution (6.7 mg/ml) are prepared in sterile 1% acetic acid and stored at 4° C. The collagen stock solution is diluted as needed in ice cold 60% ethanol, and 150 μl of the resulting solution containing 10 μg of diluted collagen is added to each well (cm2).
The collagen solution is allowed to dry at room temperature overnight (ca. 18 hours). After drying, the wells are sterilized under UV light for one hour, followed by a preincubation with cell culture medium (30 minutes). The preincubation medium is removed immediately prior to addition of cells and fresh medium. Cells are plated in the Transwells® at confluent density. The volumes of medium added will be 0.5 ml to the upper chamber and 1.0 ml to the bottom chamber. Culture medium is changed every two days. The cultures maintained in 12 well plates are allowed to differentiate a minimum of 10 days before use. The integrity of cell monolayers and formation of tight junctions is visualized by monitoring the maintenance of a slightly higher medium meniscus in the inserts as compared to the bottom wells.
Formation of tight junctions is confirmed experimentally by assay of the rate of [3H]-inulin diffusion from the top well into the bottom chamber or by measurement of transepithelial resistance across the monolayer. Transcytosis is assayed by replacement of medium, usually in the top well, with an appropriate volume of medium containing various concentrations of [125I]-labeled protein of interest. Iodination is performed according to Park et al., “Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component,” Infect. Immun. 71:1147-1154 (2003), which is hereby incorporated by reference in its entirety. Transport of radiolabeled protein is monitored by sampling the entire contents of opposite wells, which is usually the bottom wells. Aliquots (0.5 ml) of the sampled medium are filtered through a Sephadex G-25 column, and 0.5 ml fractions are collected. The amount of radioactivity in the fractions is determined in a γ-counter. The amount of transcytosed protein is normalized and expressed as fmole/hr/cm2. A minimum of two replicates per condition is included in each experiment, and experiments typically are reproduced at least three times.
Example 6
In Vivo Toxicity Assay in Mice
The toxicity of proteins of interest are bioassayed in mice. Proteins are diluted in phosphate buffered saline, including 1 mg/ml bovine serum albumin, and injected intraperitoneally (i.p.) into animals. The proteins are administered in a 100 μl aliquot of solution at concentrations of 1-100 ng per animal (average weight  ̃25 g). Any animals that survive exposure to the toxic derivatives are monitored for a total of 2 weeks to detect any non-specific toxicity.
Example 7
The BoNT Substrate-Cleavage Assay
Engineered proteins are assayed for endoprotease activity using either mouse brain synaptosomes and recombinant SNAP-25 for BoNT A and BoNT E as the source of the substrate. Native or reduced proteins are incubated with 10 to 50 μg of synaptosomal membranes in reaction buffer containing 50 mM HEPES, pH 7.1, 20 μM ZnCl2, and 1% N-octyl-β-D-glucopyranoside. Reduced protein are prepared by incubation with DTT (20 mM; 1 hr; room temperature) in phosphate buffered saline. The cleavage reaction is initiated by addition of engineered protein (200 nM final concentration) to substrate, and the reaction is allowed to proceed for 3 hours at 37° C. Endoprotease activity is assayed using Western blot analysis and anti-C-terminal SNAP-25 antibodies (StressGen) for immunodetection of substrate. For visualization of SNAP-25, samples are separated on 16.5% Tris-tricine gels. After separation, proteins are transferred to nitrocellulose membranes (Micron Separations) in Tris-glycine transfer buffer at 50 volts for 1 hr. Blotted membranes are rinsed in distilled water and stained for 1 min with 0.2% Ponceau S in 1% acetic acid. Following a brief rinse with distilled water, molecular weight markers and transferred proteins are visualized. Membranes are destained in phosphate buffered saline-Tween (pH 7.5; 0.1% Tween 20), then blocked with 5% non-fat powdered milk in phosphate buffered saline-Tween for 1 hr at room temperature. Subsequently, membranes are incubated in 0.5% milk with a 1:5,000 dilution of anti-SNAP-25 polyclonal antibody. Secondary antibody is used at 1:20,000 dilution. Membranes are washed again (5×) and visualized using enhanced chemiluminescence (SuperSignal®West Pico, Pierce) according to manufacturer's instructions. Membranes are exposed to film (Hyperfilm ECL, Amersham Biosciences) for times adequate to visualize chemiluminescence bands. Peptides are identified by comparison with known standards. The BoNT B substrate-cleavage assay is performed according to the published protocol (Caccin et al., “VAMP/Synaptobrevin Cleavage by Tetanus and Botulinum Neurotoxins is Strongly Enhanced by Acidic Liposomes,” FEBS Lett. 542:132-136 (2003), which is hereby incorporated by reference in its entirety).
Example 8
Cloning Procedures
Preparation of the DNA Template for PCR
Outlined in detail infra are the procedures used to engineer BoNT A derivatives. A similar strategy for engineering all BoNT derivatives can be carried out.
25 μg of the pure Clostridium botulinum type A (Hall strain) genomic DNA was isolated from bacterial pellet separated from the 100 ml of the culture according to Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Plainview, N.Y.: Cold Spring Harbor Laboratory Press (1989), which is hereby incorporated by reference in its entirety. DNA was precipitated and dissolved in 1×TE, pH 8.0, at concentration  ̃0.8 mg/ml.
Genomic DNA, isolated from the mixture of the anaerobic bacteria from the soil, was prepared according to the following protocol: 1000 g of the soil taken from Central Park, New York, were triturated in 2 liters of Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen, Cat. #14190-144). Crude extract was filtered through Kimwipes EX-L wipes (Kimberly-Clark, Neenah, Wis.) and concentrated on a stirred ultrafiltration cell (Millipore (Billerica, Mass.), Cat. #5123) with Ultracel 100-KDa cutoff membrane (Millipore, Cat. #14432) to a final volume of 5 ml. Four liters of cooked meat medium (Difco (Franklin Lakes, N.J.), Cat. #226720), prepared according to manufacturer's protocol were inoculated with 5 ml of concentrated soil extract. After 168-hour incubation at 37° C. without agitation or aeration, a mixture of anaerobic bacteria was separated from the supernatant by centrifugation on Sorwall GS3 rotor (7000 rpm, 25 min., 4° C.) and processed for the isolation of the total genomic DNA on Qiagen (Valencia, Calif.) Genomic tips (Cat. #10262), with additional components also purchased from Qiagen (Cat. #19060, Cat. #19133, Cat. #19101), according to manufacturer's protocol (Qiagen Genomic DNA Handbook). From the cells recovered from 4 liters of the media on ten Qiagen Genomic tips, 6 mg of the genomic DNA were isolated. DNA was precipitated and dissolved in 1×TE, pH 8.0 at concentration  ̃1 mg/ml.
Example 9
PCR Amplification of BoNT DNA
25 μg of the mixed genomic DNA or 5 μg of the pure Clostridium botulinum type A genomic DNA were used per one 100-μl PCR reaction setting. Reaction conditions were designed according to manufacturer's protocols supplied with Platinum®Pfx polymerase (Invitrogen, Cat. #11708-021). All oligonucleotides and linkers were designed according to the sequence of botulinum Neurotoxin type A cDNA obtained from Genebank (Accession #: M30196). Annealing temperatures were deduced from the structure of each set of the oligonucleotides used for the PCR.
Example 10
Engineering of Non-Expression Vector pLitBoNTA, Carrying Coding Part of BoNT A td
Plasmid encoding botulinum Neurotoxin A light chain (pLitBoNTALC) was obtained by the following protocol: The annealed phosphorylated linkers







(SEQ ID NO: 26)


CBA1: 5′-pCTAGCATGCCATTTGTTAATAAACAATTTAATTATAAG


and


 


(SEQ ID NO: 27)


CBA2: 5′-pGATCCTTATAATTAAATTGTTTATTAACAAATGGCATG






were subcloned into vector pcDNA3.1/Zeo(+) (Invitrogen, Cat. #V86020), pre-digested with the restriction endonucleases NheI and BamHI and dephosphorylated, resulting in plasmid pcDBoNTALC1. The 620 b.p. PCR product, obtained on genomic DNA as a template with the oligonucleotides










CBA03:



(SEQ ID NO: 28)



5′-TATCTGCAGGGATCCTGTAAATGGTGTTGATATTGCTT



 



ATATAAAAATTCC



 



and



 



CBA04:



(SEQ ID NO: 29)



5′-TATGAATTCACCGGTCCGCGGGATCTGTAGCAAATTT



 



GCCTGCACC






was digested with the restriction endonucleases BamHI and EcoRI and subcloned into pre-digested plasmid pcDBoNTALC1, resulting in plasmid pcDBoNTALC2. The 630 b.p. PCR product, obtained on genomic DNA as a template with the oligonucleotides










CBA05:



(SEQ ID NO: 30)



5′-TATACCGCGGTAACATTAGCACATGAACTTATACA



 



TGCTGGACATAGATTATATG



and



 



CBA06:



(SEQ ID NO: 31)



5′-CATAGAATTCAAACAATCCAGTAAAATTTTTTAGTTT



 



AGTAAAATTCATATTATTAATTTCTGTATTTTGACC,






was digested with the restriction endonucleases SacII and EcoRI and subcloned into pre-digested plasmid pcDBoNTLC2, resulting in plasmid pcDBoNTLC3. The annealed phosphorylated linkers










CBA8:



(SEQ ID NO: 32)



5′-pAAT TCTATAAGTTGCTATGTGTAAGAGGGATAAT



 



ACTAGTCACACTCAATCT



and



 



CBA9:



(SEQ ID NO: 33)



5′-pCTAGAGATTGAGTGTGACTAGTTATTATCCCTCTTA



 



CACATAGCAACTTATAG






were subcloned into vector pcDBoNTLC3, pre-digested with the restriction endonucleases EcoR and XbaI and dephosphorylated, resulting in plasmid pcDBoNTALC. The annealed phosphorylated linkers










CBA10:



(SEQ ID NO: 34)



5′-pCGCGTTAGCCATAAATCTGGTTATAAGCGCGC



 



GAGGTGTTAAGTG



and



 



CBA11:



(SEQ ID NO: 35)



5′-pCTAGCACTTAACACCTCGCGCGCTTATAACCAGA



 



TTTATGGCTAA






were subcloned into vector pLitmus38i (New England Biolabs, Cat. #N3538S), pre-digested with the restriction endonucleases MluI and NheI and dephosphorylated, resulting in plasmid pLit38iMod. The 1230 b.p. DNA fragment, isolated from the plasmid pcDBoNALC after its digest with restriction endonucleases NheI and ApaI was subcloned into pre-digested and dephosphorylated vector pLit38iMod, resulting in plasmid pLitBoNTALC.

Plasmid encoding botulinum Neurotoxin A heavy chain (pLitBoNTAHC) was obtained by the following protocol: The 1450 b.p. PCR product obtained on the genomic DNA as a template with the oligonucleotides







CBA12:


(SEQ ID NO: 36)


5′-AATCTGCAGCCACAGCTGTGGGGTACCTTAATTGGTCA


 


AGTAGATAGATTAAAAGATAAAGTTAATAATACACTTAGTACAGA


 


TATACC


and


 


CBA13:


(SEQ ID NO: 37)


5′-ATTAGGGCCCTTAATTAAGCGGCCGCCTCGAGC


 


TATTACAGTGGCCTTTCTCCCCATCCATCATCTACAGGAATAAATTC






was digested with restriction endonucleases ApaI and PstI and subcloned into pre-digested and dephosphorylated vector pLitmus38i, resulting in plasmid pLitBoNTAHC1. Two PCR products, 490 b.p., obtained on the genomic DNA as a template with the oligonucleotides










CBA14:



(SEQ ID NO: 38)



5′-ATACTGCAGTCTAGACCAAGGATACAATGACGATG



 



ATGATAAGGCA TTAAATGATTTATGTATCAAAGTTAATAATTGGG



and



 



CBA15:



(SEQ ID NO: 39)



5′-GCCTAAAAACATAGCCGCTTCGGTCGCTTTATTAACTT



 



TCTTTACATAGTCTGAAG






and 720 b.p., obtained on genomic DNA as a template with the oligonucleotides










CBA16:



(SEQ ID NO: 40)



5′-TAATAAAGCGACCGAAGCGGCTATGTTTTTAGGCT



 



GGGTAGAACAATTAG



and



 



CBA17:



(SEQ ID NO: 41)



5′-TATAGGGCCCCCTAGGGGTACCTCTATTATCATATATAT



 



ACTTTAATAATGCATCTTTAAGAC






were mixed with the molar ratio 1:1 and re-PCRed with oligonucleotides CBA14 and CBA17, resulting in 1170 b.p. PCR product, which was digested with restriction endonucleases PstI and KpnI and subcloned into pre-digested and dephosphorylated vector pLitBoNTAHC1, leading to plasmid pLitBoNTAHC.

Plasmid pLitBoNTA, encoding the entire sequence of BoNT A was obtained by ligating a 2615 b.p. DNA fragment from the vector pLitBoNTAHC, digested with restriction endonucleases XbaI and ApaI into pre-digested and dephosphorylated vector pLitBoNTALC. The size of pLitBoNTA is 6712 b.p. with 3900 b.p. of BoNT A coding sequence.
Example 11
Engineering Plasmid pETcBoNTA for the BoNT A td Expression in E. coli 
pETCBoNTA was obtained by subcloning DNA fragment obtained after the digest of pLitBoNTA vector with NheI and NotI into pre-digested and dephosphorylated expression vector pETcoco2 (Novagen (San Diego, Calif.), Cat. #71148-3) and resulted in 16,194 b.p. BoNT A td expression vector pETCBoNTA.
Example 12
Engineering Donor Plasmid pFBSecBoNTA for the Expression of BoNT A td in Insect Cells
pFBSecBoNTA was obtained by the following protocol: 112 b.p. PCR product, synthesized on plasmid pBac-3 (Novagen, Cat. #70088-3) with oligonucleotides







CBA 22:


(SEQ ID NO: 42)


5′-TAAGCGCGCAGAATTCTCTAGAAT GCCCATGTTAAGCGCTATTG


and


 


CBA23:


(SEQ ID NO: 43)


5′-TAAGCTAGCGTGATGGTGGTGATGATGGACCATGGCC






and digested with restriction endonucleases BssHII and NheI was subcloned into pre-digested and dephosphorylated vector pLitBoNTA, resulting in plasmid pLitSecBoNTA. DNA fragment, isolated from pLitSecBoNTA digested with BssHII and NotI was subcloned into pre-digested and dephosphorylated vector pFastBacTM1 (Invitrogen, Cat. #10360-014), resulting in 8764 b.p. plasmid pFBSecBoNTA.

Example 13
Engineering the BoNT A Coding Sequence to Enable Expression of Toxin Derivatives
The DNA template was obtained as either pure genomic DNA isolated from Clostridium botulinum type A cultures, or as mixed genomic DNA isolated from anaerobic bacteria of soil. BoNT A DNA was amplified by PCR using the high fidelity Platinum Pfx polymerase (Invitrogen, Carlsbad, Calif.). The full-length coding sequence of BoNT A toxic derivative (td) was obtained after consecutive subcloning of five PCR fragments and two phosphorylated linkers into the modified vector pLitmus38i (New England Biolabs, Beverly, Mass.), resulting in plasmid pLitBoNTA. This strategy was used to minimize infidelity during the PCR reaction and to enable the introduction of targeted mutations and endonuclease restriction sites for subsequent engineering of expressed toxin products.
The details of the final construct (td) in comparison with native BoNT A (wt) are shown in FIG. 3, illustrating new restriction sites, eliminated alternative translation sites, and amino acids inserted or substituted. The construct encoding full-length BoNT A td was obtained by ligation of the DNA inserts from the plasmid encoding the toxin heavy chain (“HC”) into the plasmid encoding the toxin light chain (“LC”). Plasmid encoding the LC of BoNT A td was generated by consecutive ligation of two PCR products and two phosphorylated linkers into vector pLitmus38i. It contains multiple unique restriction sites upstream from the 5′-end of the LC sequence, the unique endonuclease restriction site NheI upstream from the first methionine codon, and endonuclease restriction sites for BamHI and EcoRI introduced by silent mutations flanking the minimal catalytic domain (Kadkhodayan et al., “Cloning, Expression, and One-Step Purification of the Minimal Essential Domain of the Light Chain of Botulinum Neurotoxin Type A,” Protein Expr. Purif. 19:125-130 (2000), which is hereby incorporated by reference in its entirety) of the protein at the codons for Lys11 and Phe425. Two additional mutations encoding substitutions Lys438>His and Lys440>Gln were introduced to minimize non-specific proteolysis of the BoNT A td propeptide during expression. A unique restriction site for XbaI was introduced by silent mutation at the codon. Plasmid, encoding the HC of BoNT A td, was generated by consecutive ligation of two PCR products into the pLitmus38i vector. First, the PCR product encoding the receptor-binding domain of BoNT A was subcloned into the vector pLitmus38i. Second, the PCR product encoding the toxin's translocation domain, obtained by re-PCR of two smaller PCR products was subcloned into plasmid encoding the toxin's receptor binding domain. The final plasmid contains a unique XbaI site at the 5′-end of the coding sequence introduced by silent mutation of the codon Asp443, mutation of Lys444>Gln to minimize non-specific proteolysis of the BoNT A td propeptide, insertion of codons for four aspartic acid residues between Asn447 and Lys448 to create an enterokinase cleavage site, four silent mutations at Ala597, Thr598, Glu599, and Ala600 to inactivate the putative internal DNA regulatory element, a unique KpnI site introduced at the codon for Gly829 by silent mutagenesis, and multiple unique restriction sites at the 3′-end of the construct after the stop codon. DNA encoding the Ala597-Ala699 sequence was mutated, because it contains an internal Shine-Dalgarno sequence upstream from internal methionine codon which can result in co-translational contamination of recombinant protein expressed in E. coli (Lacy et al., “Recombinant Expression and Purification of the Botulinum Neurotoxin Type A Translocation Domain,” Protein Expr. Purif. 11:195-200 (1997), which is hereby incorporated by reference in its entirety), the initial choice for an expression system to test.
A second full-length BoNT A gene derivative was designed to render the BoNT A atoxic (ad, atoxic derivative). Using site-directed mutagenesis with two synthetic oligonucleotides, a single point mutation, E224>A, was introduced into plasmid pLitBoNTA to inactivate the proteolytic activity responsible for BoNT A neurotoxicity resulting in plasmid pLitBoNTAME224A (Kurazono et al., “Minimal Essential Domains Specifying Toxicity of the Light Chains of Tetanus Toxin and Botulinum Neurotoxin Type A,” J. Biol. Chem. 267:14721-14729 (1992); Lacy et al., “Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998); Agarwal et al., “Structural Analysis of Botulinum Neurotoxin Type E Catalytic Domain and Its Mutant Glu212>Gln Reveals the Pivotal Role of the Glu212 Carboxylate in the Catalytic Pathway,” Biochemistry 43:6637-6644 (2004), which are hereby incorporated by reference in their entirety). Atoxic derivatives produced in this way will better preserve the structural moieties responsible for toxin immunogenicity, trafficking, and cell recognition sites.
A third full-length BoNT A derivative was designed to test the utility of the genetic engineering methodology to produce fusion proteins, using GFP as an example. The sequence encoding the minimal catalytic domain of the BoNT A LC (Kadkhodayan et al., “Cloning, Expression, and One-Step Purification of the Minimal Essential Domain of the Light Chain of Botulinum Neurotoxin Type A,” Protein Expr. Purif. 19:125-130 (2000), which is hereby incorporated by reference in its entirety) was excised from plasmid pLitBoNTA and replaced with a GFP-coding sequence to create plasmid pLitGFPBoNTAHC encoding a GFP derivative of BoNT A (gfpd). The GFP-encoding sequence was obtained by PCR with two synthetic oligonucleotides on the plasmid pEGFP-N3 (Clontech, Palo Alto, Calif.). The fusion protein was specifically designed to preserve structural features responsible for cell binding and intracellular trafficking.
All intermediate DNA constructs as well as plasmids pLitBoNTA, pLitBoNTAME224A, and pLitGFPBoNTAHC were checked by multiple restriction digests. pLitBoNTA and pLitBoNTAME224A were sequenced with twelve BoNT A-specific synthetic oligonucleotides, while pLitGFPBoNTAHC was sequenced with ten BoNT A-specific synthetic oligonucleotides and two GFP-specific oligonucleotides as primers, resulting in a set of overlapping sequences which covered all coding parts of all of the above plasmids. All sequences were demonstrated to be free of unexpected mutations.
Example 14
Expression of the Recombinant BoNT A Derivatives in E. coli 
Expression plasmids were transfected into E. coli Rosetta-gami B (DE3) competent cells (Novagen, Cat. #71136-3) by the heat-shock method according to manufacturer protocol. Bacterial cultures were grown in LB media containing 50 mg/l carbenicillin, 15 mg/l kanamycin, 12.5 mg/l tetracycline and 34 mg/l chloramphenicol. Various conditions, affecting the plasmid copy number per cell without and with addition of L-arabinose (0.01% final concentration) to the bacterial medium were tested. All bacterial cultures used for protein expression were grown at 37° C. until reaching OD@600 nm  ̃0.3-0.4. Prior to the induction of the expression, bacterial cultures were split to test influence of the temperature on the yield and quality of the expressed product. Upon induction (OD@600 nm  ̃0.5-0.7), cultures were grown at 37° C., 25° C., and 12° C. Final IPTG concentration in the growth medium used for induction was 0.5 mM. For the time-course study, samples of the culture at 1, 3, 6, 9, and 12 hours after induction were collected and analyzed. Under the optimal conditions the E. coli cultures were incubated overnight in the presence of L-arabinose at 37° C. until reaching OD  ̃0.4@600 nm. The temperature of the bacterial suspensions was then lowered to 12° C. over one hour, and IPTG was added to a final concentration of 0.5 mM. After induction, culture growth was allowed to continue in a shaker incubator at 12° C. for six more hours. The bacterial pellet was then harvested by centrifugation on Sorwall GS3 rotor (7000 rpm, 25 min., 4° C.) and processed for recombinant protein isolation. Cells kept on ice were resuspended in BugBuster lysis reagent (Novagen, Cat. #70584-4) with the volume ratio cell paste:BugBuster solubilization reagent 1:5. The nucleic acid degradation reagent benzonaze was used instead of the mixture sonication (Novagen, Cat. #70746-3), 1000 U/ml final concentration, recombinant lysozyme (Novagen, Cat. #71110-4), 50 U/ml final concentration, and a cocktail of protease inhibitors “Complete” (Roche (Switzerland), Cat. #1697498), 1 tablet/50 ml final concentration were added simultaneously to the paste in the process of resuspension. Approximately 30 minutes after resuspension, the non-viscous lysate was cleared by centrifugation on Sorwall SS34 rotor (17000 rpm, 25 min, 4° C.) and processed for the further protein purification.
An E. coli expression system was the first tested for a number of reasons. First, other laboratories have reported expression of recombinant partial length BoNT A domains in this system (Rigoni et al., “Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun. 288:1231-1237 (2001); Chaddock et al., “Expression and Purification of Catalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridium Botulinum Toxin Type A,” Protein Expr. Purif. 25:219-228 (2002); Lalli et al., “Functional Characterization of Tetanus and Botulinum Neurotoxins Binding Domains,” J. Cell Sci. 112:2715-2724 (1999); Kadkhodayan et al., “Cloning, Expression, and One-Step Purification of the Minimal Essential Domain of the Light Chain of Botulinum Neurotoxin Type A,” Protein Expr. Purif. 19:125-130 (2000); Lacy et al., “Recombinant Expression and Purification of the Botulinum Neurotoxin Type A Translocation Domain,” Protein Expr. Purif. 11:195-200 (1997), which are hereby incorporated by reference in their entirety). A second reason for selecting an E. coli expression system is that many recombinant proteins can be expressed in E. coli with good yield and stability. A third reason is that non-canonical E. coli codons in the BoNT A sequence can be overcome by utilizing a bacterial strain carrying a plasmid encoding tRNA for the rare codons. A fourth reason is that toxicity of the full-length BoNT A to the host can be minimized by using an expression plasmid that allows regulation of the transition from low to medium plasmid copy numbers. Fifth, proper disulfide bridge formation in the recombinant proteins can be optimized by utilizing an E. coli strain with trxB− gor− mutations. Sixth, degradation of recombinant proteins in the host can be minimized by utilizing an E. coli Rosetta-gami strain with lon− ompT− mutations, in which two major proteolytic enzymes are inactivated.
Expression plasmids were obtained by single-step subcloning of the coding portion of BoNT A derivatives—td, ad, and gfpd into the expression vector pETcoco2 (Novagen, San Diego, Calif.). The resulting constructs contain DNA, encoding sequence MHHHHHHGAS . . . (SEQ ID NO: 44) and flanked with NheI unique restriction site in front of the first native methionine codon. The pETcoco system combines the advantages of T7 promoter-driven protein expression with the ability to control plasmid copy number. The pETcoco vectors are normally maintained at one copy per cell. In the single-copy state, pETcoco clones are extremely stable, which is especially important for target genes that are toxic to the host. Copy number can be amplified to 20-50 copies per cell by the addition of L-arabinose to the culture medium. The pETcoco vectors in λDE3 lysogenic hosts can be induced to increase expression of the target gene by as much as 2,500-fold over background when IPTG is added to the culture media. A 6-His tag was added to each recombinant protein to enable affinity purification. The affinity tag was added to the N-terminus, because prior studies found that addition of an affinity tag to the C-terminus results in loss of the toxin's physiological activity (Shapiro et al., “Identification of a Ganglioside Recognition Domain of Tetanus Toxin Using a Novel Ganglioside Photoaffinity Ligand,” J. Biol. Chem. 272:30380-30386 (1997), which is hereby incorporated by reference in its entirety), while adding a hexahistidine tag to the N-terminus allowed expression and purification of the light chain domain with retained enzymatic activity (Kadkhodayan et al., “Cloning, Expression, and One-Step Purification of the Minimal Essential Domain of the Light Chain of Botulinum Neurotoxin Type A,” Protein Expr. Purif. 19:125-130 (2000), which is hereby incorporated by reference in its entirety).
All expression constructs were transformed into E. coli Rosetta-gami B (DE3) competent cells (Novagen) and were grown in LB media containing ampicillin, kanamycin, tetracycline, and chloramphenicol. Ampicillin was added to select for colonies carrying pETcoco derived bla marker, kanamycin and tetracyclin were added to select for thioredoxin (trxB) and glutathione reductase (gor) mutations, thus improving the chances for proper disulfide bond formation in the E. coli cytoplasm (Derman et al., “Mutations that Allow Disulfide Bond Formation in the Cytoplasm of Escherichia Coli,” Science 262:1744-1747 (1993); Prinz et al., “The Role of the Thioredoxin and Glutaredoxin Pathways in Reducing Protein Disulfide Bonds in the Escherichia Coli Cytoplasm,” J. Biol. Chem. 272:15661-15667 (1997), which are hereby incorporated by reference in their entirety). Chloramphenicol was added to the medium to select for colonies containing helper plasmids that provide tRNAs for rare codons, thereby increasing the expression of proteins such as BoNT A encoded by DNA with codons non-canonical for E. coli. 
Multiple conditions were tested to optimize expression of the BoNT A full length derivatives. Cultures were grown with and without L-arabinose in the media, and different IPTG concentrations were evaluated for induction. Incubation temperatures and time were also optimized for BoNT derivative expression. Under optimal conditions, the E. coli cultures were incubated overnight in the presence of L-arabinose at 37° C. until reaching OD  ̃0.4 at 600 nm. The temperature of the bacterial suspensions was then lowered to 12° C. over one hour, and IPTG was added to a final concentration 0.5 mM. After induction, culture growth was allowed to continue in a shaker incubator at 12° C. for six more hours. The bacterial pellet was then harvested by centrifugation, lysed with BugBuster lysis reagent (Novagen) in the presence of nucleic acid degradation reagent benzonaze (Novagen), lysozyme, and a cocktail of protease inhibitors. The lysate was cleared by centrifugation and purified by incubation with a Ni-NTA affinity resin. The supernatant and eluate from the Ni-NTA agarose were run on 8% SDS PAGE gels, and analyzed by Western blotting with polyclonal antibodies raised against the full-length BoNT A inactivated toxioid. Rosetta-gami B (DE3) E. coli transformed with the empty vector was used as the negative control. Native BoNT A in SDS-PAGE loading buffer was used as the positive control.
FIG. 4 illustrates the results of E. coli expression and purification protocols for BoNT A td. The expressed protein was soluble and could be purified using the chelate affinity tag. However, the molecular weight of the recombinant BoNT A td full length propeptide expressed was significantly lower than that of the native full-length BoNT A propeptide. Extensive proteolysis was observed with all purification and expression protocols tested in E. coli, even when the toxin derivatives were expressed by the cells in the single-copy plasmid state. This instability may be related to the systems available in E. coli for post-translational processing of proteins, with improper folding and disulfide bonding making the recombinant toxins susceptible to degradation. Similar results were obtained when attempting to express the atoxic (ad) and GFP-(gfpd) derivatives of BoNT A in E. coli. The problems encountered with E. coli based expression systems with respect to native protein folding and extensive proteolysis of the recombinant product, may be resolved by modification and optimization of the E. coli expression system.
Example 15
Expression of BoNT A Derivatives in Baculovirus-Based System
Bac-to-Bac® baculovirus expression system (Invitrogen, Cat. #10359-016) was used for the generation of the recombinant baculoviruses. A protocol for the insect cell culture was taken from the manual supplied with the kit. Recombinant donor plasmids were transformed into Max Efficiency DH10BacTM competent cells (Invitrogen, Cat. #10361-012). Colonies containing recombinant bacmid were identified by disruption of the lacZa gene and selected by the absence of developing blue color, while growing on the plate with the chromogenic substrate Bluo-gal (Invitrogen, Cat. #15519-028). High molecular weight DNA was isolated from the selected colonies on DNA plasmid purification system (Qiagen, Cat. #12245). Transposition of the DNA of interest into baculovirus genome was confirmed by PCR on high molecular weight DNA with oligonucleotides CBA14 and CBA17, resulting in amplification of 1170 b.p. DNA band in samples where transposition took place. Bacmids were used to transfect serum-free medium adapted Sf9 insect cells (Invitrogen, Cat. #11496-015) to produce baculoviruses. Transfection was performed by the following protocol: 9×105 cells were seeded per one 35-mm well in 2 ml of unsupplemented Grace's insect cell culture medium (Invitrogen, Cat. #11595-030). Cells from a 3 to 4 day-old suspension culture in mid-log phase with a viability of >97% were used for experiment. Cells were attached to the plastic at 27° C. for at least one hour in advance and transfected with the lipophylic complexes, formed after mixing bacmid with Cellfectin® transfection reagent (Invitrogen, Cat. #10362-010), according to the protocol supplied by the manufacturer. 72 hours after transfection, the supernatant containing recombinant baculoviruses was harvested and separated from the cells by low-speed centrifugation (Sorwall GS 3 Rotor, 2000 rpm, 20 min, 4° C.). The supernatant represents the primary baculoviral stock. Amplification of this baculoviral stock and viral plaque assay was performed according to the protocol supplied by the manufacturer. Experiments related to identification of the optimal MOI and time-course studies of recombinant protein expression were also performed according to the manufacturer recommendations.
For the purpose of protein expression, Sf9 cells were grown as a shaken culture in a SF900 II serum-free medium (Invitrogen, Cat. #10902-088) at 27° C. in humidified atmosphere. At the density of the cell culture  ̃1.2×106/ml, baculovirus stock in the same medium was added to suspension at MOI  ̃0.1. Incubation continues for another  ̃50 hours, after which medium was separated from the cells by centrifugation (Sorwall GS 3 Rotor, 2000 rpm, 20 min, 4° C.) and further processed for the protein purification by the procedure outlined below. Sf9 cells are very sensitive to growth conditions. They require a constant temperature of 27±1° C., good aeration of shaking cultures, and a sterile environment. If ambient temperatures rise above 27° C., refrigeration is required in the incubator used. An incubator sufficiently large to produce sufficient quantities of BoNT derivatives for biological testing is recommended.
To avoid poisoning of the insect cell host, the BoNT A td construct was modified by adding a signal peptide to provide for secretion of the recombinant proteins to the medium. Targeting the recombinant toxins for secretion also resulted in proper disulfide bond formation between the toxin's light and heavy chains. Improvements to this expression system were tested as described infra.
To increase the total yield of the recombinant protein, donor recombinant baculovirus plasmids and bacmids were generated with an expression cassette that allows expression of the recombinant protein to be driven by two separate and independent promoters simultaneously, p10 and PH (donor plasmid pFastBacTM Dual, Invitrogen, Cat. #10712-024).
To stabilize and increase the titer of the recombinant baculoviral stock, an approach outlined in BaculoDirect® Expression System protocol (Invitrogen (Carlsbad, Calif.), Cat. #12562-021) was used that allows negative selection to remove non-recombinant baculovirus that tend to appear in amplified stocks over the time. To improve purification of the toxins, the affinity of the recombinantly expressed proteins for Ni-NTA resin was increased by generating additional BoNT constructs with longer N-terminal His tags.
The advantages of a baculovirus expression system include proper disulfide bridge formation which has been demonstrated for numerous recombinant proteins in this system; protein purification, which is facilitated when serum-free culture medium is utilized and the expressed proteins contain a short secretory signal and affinity tag; physiological activity similar to native progenitors can be retained in the expressed products; and the absence of endotoxins endogenous to E. coli, which facilitates biological testing and therapeutic use of the expressed proteins (Allen et al., “Recombinant Human Nerve Growth Factor for Clinical Trials: Protein Expression, Purification, Stability and Characterisation of Binding to Infusion Pumps,” J. Biochem. Biophys. Methods. 47:239-255 (2001); Curtis et al., “Insect Cell Production of a Secreted form of Human Alpha(1)-Proteinase Inhibitor as a Bifunctional Protein which Inhibits Neutrophil Elastase and has Growth Factor-Like Activities,” J. Biotechnol. 93:35-44 (2002), which are hereby incorporated by reference in their entirety). The disadvantages of this system are its cost, time-consuming procedures, and generally the yield of proteins is not as high as in E. coli. Furthermore, because the regulated exocytosis machinery is well preserved across eukaryotic species from yeast to mammals, expression of BoNT A in this system can potentially lead to the host poisoning and cellular death. Nonetheless, since Clostridial neurotoxins are known to pass through epithelial cells by transcytosis without any toxic affects (Simpson, “Identification of the Major Steps in Botulinum Toxin Action,” Annu. Rev. Pharmacol. Toxicol. 44:167-193 (2004); Park et al., “Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component,” Infect. Immun. 71:1147-1154 (2003), which are hereby incorporated by reference in their entirety), and the toxin constructs described herein are designed to remain in the single-chain propeptide form until processed to dichain mature form by enterokinase, this system is worth further testing.
Plasmid constructs for expression of BoNT A derivatives in this system were subcloned into the donor vector pFastBacTM1 (Invitrogen). To facilitate secretion of the recombinant proteins into the media and to allow purification of the recombinant proteins on Ni-NTA agarose, a DNA sequence coding the gp64 signal peptide and a hexahistidine affinity tag MPMLSAIVLYVLLAAAAHSAFAAMVHHHHHHSAS . . . (SEQ ID NO: 45), flanked with unique NheI restriction site in front of the first native methionine codon, was introduced by cloning of PCR product into all constructs. FIG. 5 provides a schematic representation of the BoNT A derivatives targeted for expression in the baculovirus system. The signal peptide shown in the illustrated recombinant proteins is removed by secretase processing during intracellular trafficking (von Heijne, “Signals for Protein Targeting Into and Across Membranes,” Subcell. Biochem. 22:1-19 (1994), which is hereby incorporated by reference in its entirety). Expression of the genes in the vector pFastBacTM1 is controlled by the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedrin (PH) promoter. Recombinant donor plasmids were transformed into DH10Bac® E. coli competent cells. Once the pFastBacTM based expression plasmid is in cellular cytoplasm, transposition occurs between the arms of mini-Tn7 element flanking the expression cassette in pFastBacTM based vector and the mini-attTn7 target site on the baculovirus shuttle vector (bacmid), already present in the cells. This event generates a recombinant bacmid. Transposition requires additional proteins supplied by helper plasmids also present in competent cells. Selection of the recombinant bacmid clones was performed visually (by size and color). The molecular nature of the isolated DNAs was confirmed by PCR with the specific oligonucleotide primers.
Recombinant bacmids and negative control bacmids (obtained as a result of transposition with empty donor plasmids) were transfected into Sf9 insect cells with the lipophilic reagent Cellfectin (Invitrogen). After 96 hours the recombinant baculoviral stock was harvested and used for infection of freshly seeded Sf9 cells.
Secondary baculoviral stock was used for multiple purposes, which include, testing the expression of recombinant proteins, amplifying recombinant baculoviruses and generating tertiary stock for future use, calculating the titer of recombinant baculoviruses, identifying the optimal ratio for multiplicity of infection (MOI), and establishing the optimal time course for protein expression. Baculovirus titer was calculated for each newly amplified baculoviral stock. For all BoNT A constructs tested, the optimal MOI was found to be  ̃0.1 pfu per cell and the optimal time for the protein harvest was found to be  ̃50 hours after infection. When recombinant proteins were allowed to accumulate in the media for 72 hours or longer, a significant portion of the recombinant protein was degraded due to virus-induced cellular lysis.
FIG. 6 illustrates the protein expression results for the toxic (td), atoxic (ad), and GFP-linked (gfpd) full-length propeptide derivatives of BoNT A (cultures harvested at 50 hrs). All recombinant proteins were soluble and secreted into the media, could be purified by binding to the affinity resin, and have the expected mobility on SDS PAGE comparable to the mobility of single chain wt BoNT A. The recombinant BoNT A derivatives expressed using these conditions were free of degradation products recognized by the polyclonal antibody.
Example 16
Enterokinase Processing and LC-HC Disulfide Bridges
FIG. 7 illustrates a dosage-titration curve for cleavage of the propeptide constructs with recombinant enterokinase (rEK), using the td derivative as an example. For the processing of the single-chain (SC) protein, different amounts have been applied to the BoNT A td. Using 0.5 U of the enzyme at 20° C. for 8 hours was found to completely digest 1 μg of the sc BoNT A td.
To evaluate whether the disulfide bridges between the light and heavy chains of the recombinant proteins were properly formed, the recombinant propeptide derivatives were compared on reducing and non-reducing gels after digestion with excess rEK. Western blots were probed with polyclonal antibodies raised against native full-length BoNT A toxoid. The results of this experiment, shown in FIGS. 8A and 8B, demonstrate that all of the recombinant propeptides were processed into a two-subunit form by rEK, and that the subunits could be readily separated after reduction of the disulfide bridges, as expected.
Expression of a GFP-linked derivative of BoNT A is demonstrated by the green fluorescence of Sf9 cells 12 hours after infection with the recombinant baculovirus expressing BoNT A gfpd (FIGS. 8C and 8D). The significant difference in the background of FIG. 8C (recombinant baculovirus expressing BoNT A gfpd with secretion signal) versus FIG. 8D (recombinant baculovirus expressing GFP control), is believed to result from the secretion of the fluorescent recombinant protein into the media.
Example 17
Purification of the Recombinant BoNT A Derivatives
Methods to purify reasonable quantities of the full-length BoNT A derivatives were developed using BoNT A td as an example. Though the recombinant protein was found to bind to Ni-NTA resin (FIG. 5), the affinity was not sufficient to establish a one step purification scheme. The protein bound to the Ni-NTA resin in 5 mM imidazole, but was eluted from the affinity column by 40 mM imidazole. At this concentration of imidazole, there are other proteins present in the eluate, and therefore additional steps are needed to separate recombinant protein from contaminants.
Similar results were observed with the minimal essential domain of BoNT A expressed and purified in E. coli (Kadkhodayan et al., “Cloning, Expression, and One-Step Purification of the Minimal Essential Domain of the Light Chain of Botulinum Neurotoxin Type A,” Protein Expr. Purif. 19:125-130 (2000), which is hereby incorporated by reference in its entirety). The stable minimal essential domain of the LC expressed with two 6-His tags on the N- and C-termini of the protein was eluted from the Ni-NTA column by 90 mM imidazole, still a relatively low concentration of affinity eluant. Poor accessibility of the affinity tags may explain these difficulties. Interestingly, there were two fractions of the same protein from the affinity column, with the second fraction eluted in 250 mM imidazole. While 90 mM imidazole fraction was enzymatically active, as was shown in SNAP-25 peptide cleavage assay, the higher concentration imidazole eluate was not. Denaturation of the protein may explain its absence of activity and high affinity for the chelate matrix.
A multi-step protocol was developed for purifying BoNT A td to homogeneity. Sf9 cells (viable cells count before infection  ̃1.2·106/ml) grown at 27° C. in SF900II serum-free media in humidified atmosphere at 125 rpm in shaking culture were harvested and separated from the medium. At  ̃50 hours after infection with recombinant baculovirus (MOI ̃0.1), the medium was collected, precipitated with ammonium sulfate or concentrated, dialyzed, and subjected to sequential DEAE-sepharose chromatography, MonoS chromatography, Ni-NTA affinity chromatography, and FPLC-based gel filtration chromatography. FIG. 9 illustrates the results of protein purification. The yield of the pure recombinant protein was 0.35 mg from one liter of serum-free medium. The pure protein eluted from the final gel-filtration column was competent for further processing with rEK. After the rEK cleavage, chloride ions containing buffer need to be substituted with phosphate or HEPES-based buffer to avoid instability of the recombinant toxin derivatives.
Approximately 2 ml of supernatant or cleared lysate was concentrated on Amicon Ultra-4 centrifugal filter device (Millipore, Cat. #UFC803024) to  ̃1 ml. The concentration procedure was done in parallel with multiple rounds of buffer substitution aimed at removing substances which could contribute to Ni2+-stripping from the affinity resin. Final buffer composition was equal to the Ni-NTA Equilibration Buffer (infra). 20 μl of Ni-NTA suspension equilibrated in the Ni-NTA Equilibration Buffer (1:1 v/v) was added to the sample, followed by the sample incubation on the rotating platform for 1 hour. After incubation, affinity matrix was separated from the supernatant by centrifugation (3000 g, 1 min), and washed three times with Ni-NTA Equilibration Buffer, followed by centrifugation. The washing buffer was aspirated and the resin was resuspended in  ̃200 μl of SDS-PAGE loading buffer. The liquid was used for the further analysis by SDS PAGE and Western blotting.







TABLE 1





BoNT A td Purification


Composition of the buffers used:















DEAE Sepharose Equilibration Buffer: 20 mM NaH2PO4,


1 mM EDTA, pH 8.0


DEAE Sepharose Wash Buffer: 50 mM NaCl, 20 mM NaH2PO4, pH 8.0


DEAE Sepharose Elution Buffer:


500 mM NaCl, 20 mM NaH2PO4, pH 8.0


Mono S Equilibration Buffer: 20 mM NaH2PO4, pH 6.8


Mono S Wash Buffer: 25 mM NaCl, 20 mM NaH2PO4, pH 6.8


Mono S Elution Buffer: 300 mM NaCl, 20 mM NaH2PO4, pH 6.8


Ni-NTA Equilibration Buffer: 5 mM imidazole,


50 mM NaH2PO4, 300 mM NaCl, pH 8.0


Ni-NTA Wash Buffer I:


Same as above but made up with 10 mM imidazole


Ni-NTA Wash Buffer II:


Same as above but made up with 20 mM imidazole


Ni-NTA Elution Buffer:


Same as above but made up with 60 mM imidazole


HiLoad 16/60 Superdex 200PG Equilibration Buffer:


50 mM NaCl, 20 mM Tris-HCl, pH 7.5









All concentration, dialysis, and chromatography steps were performed at 4° C. 300 ml of the conditioned insect medium was either concentrated on the stirred ultrafiltration cell (Millipore, Cat. #5123) with Ultracel 100-KDa cutoff membrane (Millipore, Cat. #14432) to the final volume 5 ml, or the total protein from the medium was precipitated by addition of ammonium sulfate (60 g/100 ml) with slow stirring. Pellet was separated from the supernatant by centrifugation (5000 g, 20 min, 4° C.) and dissolved in 5 ml of DEAE-Sepharose Equilibration Buffer. Recombinant protein recovered from the first procedure was less denatured, and this procedure is preferable for future work. Scale-up production of the BoNT derivatives for biological testing could be accomplished with Tangential Flow Concentration System (Pellicon 2, Millipore, Cat. #XX42PLK60) which would enable large volumes of the media to be processed. During membrane concentration or ammonium sulfate precipitation, an insoluble precipitate forms from the pluronic surfactant included in the SF900 II media (to prevent cellular aggregation and to reduce shearing forces, thereby improving the stability of the Sf9 insect cells). The insoluble pluronic pellet was removed by centrifugation of the concentrate/ammonium sulfate precipitate (5000 g, 20 min, 4° C.), and recombinant toxin in the pellet was recovered by extracting twice with DEAE-Sepharose Equilibration Buffer, followed by centrifugation.
Recovered combined supernatant was dialyzed against 100× volumes of DEAE-Sepharose Equilibration Buffer for 16 hours, separated from the residual pellet by centrifugation, and loaded on a column (1.5×10 cm) packed with DEAE-Sepharose Fast Flow (Amersham Biosciences, Cat. #17-0709-01) pre-equilibrated in the same buffer at a buffer flow rate of 0.5 ml/min. The column was washed with  ̃15 volumes of DEAE Sepharose Wash Buffer and then a linear gradient of 100 ml DEAE Sepharose Wash Buffer: 100 ml DEAE Sepharose Elution Buffer was applied. 4-ml fractions were collected and their content was analyzed by PAGE and Western blotting. Fractions containing recombinant protein were combined and dialyzed against 100× volumes of the Mono S Equilibration Buffer for 16 hours. Resulting combined dialyzate was cleared by centrifugation and loaded at 1 ml/min on MonoS 5/50 GL FPLC column (Amersham Biosciences, Cat. #17-5168-01), pre-equilibrated in the same buffer. Column was washed with 100 ml of Mono S Wash Buffer and the linear gradient of 100 ml Mono S Wash Buffer: 100 ml Mono S Elution Buffer was applied. 2-ml fractions were collected and their content was analyzed by PAGE and Western blotting.
The fractions containing recombinant protein were combined, concentrated on the stirred ultrafiltration cell with Ultracel 100-KDa cutoff membrane to the final volume of 20 ml with sequential buffer change to Ni-NTA Equilibration Buffer. Combined fractions were loaded on a 1×4 cm column with Ni-NTA affinity resin (Novagen, Cat. #70666) pre-equilibrated in the same buffer at a buffer flow rate of 1 ml/min. The column was sequentially washed with 100 ml of Ni-NTA Wash Buffer I, followed by 100 ml of Ni-NTA Wash Buffer II, and protein was eluted from the column by 50 ml of Ni-NTA Elution Buffer. All fractions were analyzed by PAGE and Western blotting. Elution fractions enriched in recombinant protein were concentrated sequentially on the stirred ultrafiltration cell with Ultracel 100-KDa cutoff membrane followed by Amicon Ultra-4 centrifugal filter device (Millipore, Cat. #UFC803024) to a final volume of 1 ml and loaded on the FPLC HiLoad 16/60 Superdex 200PG gel filtration column (Amersham Biosciences, Cat. #17-1069-01), equilibrated with HiLoad 16/60 Superdex 200PG Equilibration Buffer. The buffer flow rate was 1 ml/min and 1-ml fractions from the column were collected and analyzed by PAGE and Western blotting.
The multi-step protocol developed for BoNT A td purification provided a yield  ̃0.35 mg of pure protein per liter of serum-free medium. It is believed that this procedure can be optimized to provide yields in the range of 0.7-0.9 mg/l. Several reasons may explain the relatively low yield in the purification of BoNT A td: 1) Significant amounts of the recombinant toxin may be lost due to non-specific adsorption to the brand-new separation media; 2) The delays which occurred between purification steps may have resulted in degradation of recombinant toxins. These delays were impossible to avoid during the initial purification attempts, because it was necessary to analyze the recombinant products before proceeding to the next purification step. The following modifications, aimed at simplifying and improving the current purification scheme, were tested.
Example 18
Biological Testing of the Recombinant BoNT A Derivatives
Two types of experiments were performed to assess whether the recombinant toxins retained the biological activities of native toxin. These were performed using the BoNT A td derivative, which was produced in sufficient quantities for biological testing. In the first test, recombinant BoNT A td was administered to mice by the intravenous route ( ̃1 ng per mouse) and the time-to-death was monitored. Death was observed approximately 12 minutes after injection. Prior symptoms of muscular weakness or paralysis were not obvious. In the second test, recombinant BoNT A td was added to mouse phrenic nerve-hemidiaphragm preparations, and its ability to inhibit acetylcholine release evoked by stimulation of the nerve trunk (0.2 Hz) was evaluated by monitoring muscle twitch. At a concentration of  ̃1×10−11 M, recombinant BoNT A td produced neuromuscular blockade in 167±17 min (n=4). To insure that the blockade could be attributed to a botulinum toxin-type action, a final experiment was done, in which the polypeptide was pre-incubated (room temperature, 60 min) with rabbit antiserum raised against the carboxy terminal half of the native BoNT A heavy chain (i.e. receptor-binding domain). In these experiments (n=3), there was no neuromuscular blockade, even when the tissues were monitored for ca. 400 minutes. The pharmaceutical preparation marketed by Allergan Inc., as “BoTox” produces neuromuscular blockade in 100 minutes at a concentration of approximately 1×10−11 M (60 Units per ml). The BoNT A, B, and G recombinant products produced by Rummel (supra) require 60 to 1000 times more BoNT to effect neuromuscular blockade in a similar timeframe.
Example 19
Preparation and Modification of the BoNT Gene Constructs
DNA and protein sequences for Clostridial toxins are accessed from the Gene bank. Constructs encoding full-length toxins are available from a number of laboratories. These known sequences and constructs provide an efficient starting point for the planned genetic manipulations.
The first type of mutation introduced is designed to improve toxin stability by site-directed mutations of low specificity protease-sensitive residues within the light-heavy chain junction region, thereby reducing susceptibility to non-specific activation and poisoning of the host organism. The second type of mutation will be introduced to create a highly specific enterokinase cleavage site between the light and heavy chains, thereby enabling external control of the cleavage event leading to toxin maturation. The third type of mutation to be introduced is designed to silently inactivate DNA elements affecting RNA transcription and protein expression in the system of choice. The fourth type of modification is designed to introduce unique restriction sites that enable easy manipulation of the toxin gene, its protein products, and chimeric proteins which may be created as required.
The modified BoNT A constructs used to produce the BoNT A toxic derivative (td) described infra demonstrates the feasibility of these methods. The objective is to determine how to best adapt the methods developed for BoNT A to producing other Clostridial neurotoxins, and in the process optimize the methodology and create a library of toxin derivatives with customized biological properties. Molecular cloning techniques are generally known in the art, and other full-length neurotoxins have successfully been cloned (Ichtchenko et al., “Alpha-Latrotoxin Action Probed with Recombinant Toxin: Receptors Recruit Alpha-Latrotoxin but do not Transduce an Exocytotic Signal,” EMBO J. 17:6188-6199 (1998), which is hereby incorporated by reference in its entirety).
Example 20
Creation and Expression of Recombinant BoNT Molecules Minimally Modified to Eliminate Toxicity
To create atoxic derivatives (“ad”) that most closely resemble the native toxin with respect to their structure and physiologic activity, a single amino acid point mutation is introduced into the active site of the toxin's metalloprotease catalytic domain. Though most toxin features in this molecule remain the same as in the native toxin, it is devoid of toxicity, because it is unable to cleave its substrate in the synaptic exocytosis machinery. The atoxic derivatives thus created are superior to other BoNT preparations being developed as vaccines, because of their structural similarity to native toxin, and their ability to generate an immune response at diverse sites along the native toxin's absorption and trafficking route. Because these derivatives are likely to compete with native toxin for the same binding sites and trafficking pathways, they may also be superior to antibody preparations as antidotes to BoNT poisoning.
The cloning and expression strategies developed can be duplicated as closely as possible in applying the methods to BoNT B and E, thereby minimizing the possibility of creating significant molecular alterations in the atoxic derivatives which might decrease their therapeutic potential. The validity of this assumption is demonstrated supra with the BoNT A atoxic derivative (ad), which has been shown to be essentially identical to native BoNT A with respect to expression level, antibody recognition, disulfide bonding, cleavage with enterokinase, and binding to Ni-NTA affinity resin.
An outline of the steps necessary to produce atoxic derivatives of BoNT B and E is as follows. Constructs encoding the atoxic derivatives (ad) of BoNT B and E are obtained by site-directed mutagenesis of BoNT B and BoNT E td constructs, using procedures established for BoNT A ad, as detailed supra. Expression constructs for BoNT B ad and BoNT E ad in the different expression systems to be tested are prepared using a protocol similar to that established for BoNT A ad, as detailed supra. The expression system, purification protocol, and rEK-cleavage protocol for BoNT B and E ad replicate the optimized procedure developed for BoNT A td and ad, as outlined supra. The expression and purification system chosen to produce the atoxic derivatives is based on the quality and yield produced by the expression systems tested.
The atoxic derivatives are tested in a substrate cleavage assay using SNAP 25 or VAMP as substrates. Though no residual proteolytic activity in the single-amino acid mutated atoxic derivatives is expected, if the rate of substrate hydrolysis for any particular atoxic derivative is significantly higher than zero, a second amino acid residue, corresponding to His227 in BoNT A, is mutated at the toxin's active site before proceeding for its further biological and functional characterization.
Prophetic Example 21
Preparation of DNA Starting Material for BoNT Serotypes B and E
DNA template for all BoNT serotypes for PCR amplification can be obtained from either pure Clostridium cultures (serotype-specific) or soil-derived anaerobic cultures from which mixed genomic DNA as a starting material may be prepared. High fidelity Platinum®Pfx polymerase is used for all PCR reactions to minimize amplification errors. BoNT B and E serotypes are described in subsequent examples.
Prophetic Example 22
Constructs for BoNT B and E
A set of oligonucleotides similar to those used for obtaining the full-length coding sequence of BoNT A td may be designed for BoNT B and E, using sequences available from Gene bank (accession number M81186 for BoNT B and X62683 for BoNT E). Sequences are carefully evaluated for unwanted DNA regulatory elements and other features that could affect protein expression in E. coli, baculovirus, and Pichia pastoris expression systems, and such elements eliminated by silent site-directed mutagenesis. Additional mutations targeted to remove low-specificity proteolysis sites in the toxin's LC-HC junction are introduced, and to introduce an enterokinase cleavage site in the LC-HC junction region. Based on the toxin sequence alignments and domain structure illustrated in FIGS. 1-3, gene regions which can be modified without affecting the recombinant toxin's biological properties are identified, and Nhe I, XbaI, KpnI, and XhoI restriction sites are introduced, similar to the design scheme executed for BoNT A td. If such mutations are impossible to make through silent mutagenesis, restriction sites are introduced via neutral amino acid insertion into structurally flexible portions of the protein sequence. Any redundant restriction sites created are eliminated by silent site-directed mutagenesis. BoNT DNA sequences that can cause premature termination of gene transcription in the expression systems or interfere with the protein expression are also modified. Expression in Pichia pastoris (Henikoff et al., “Sequences Responsible for Transcription Termination on a Gene Segment in Saccharomyces Cerevisiae,” Mol. Cell Biol. 4:1515-1520 (1984); Irniger et al., “Different Classes of Polyadenylation Sites in the Yeast Saccharomyces Cerevisiae,” Mol. Cell Biol. 11:3060-3069 (1991); Scorer et al., “The Intracellular Production and Secretion of HIV-1 Envelope Protein in the Methylotrophic Yeast Pichia Pastoris,” Gene 136:111-119 (1993), which are hereby incorporated by reference in their entirety) requires the elimination of such sequences by designing a set of PCR oligonucleotide primers which can suppress premature termination of transcription from AT-rich templates. These procedures produce constructs containing modified coding sequences for BoNT B and BoNT E td, which are used in subsequent expression studies.
Endonuclease restriction digests are used to check all intermediate DNA products. The final full-length DNA is sequenced to prove absence of unwanted mutations.
Molecular biocomputing software, supplied by the DNAstar is used to analyze and compare DNA and protein sequences. This will also optimize the creation of synthetic oligonucleotides and optimal reaction conditions for all reactions of PCR amplification.
Prophetic Example 23
Expression, Purification, and Biochemical Analysis of Toxic Derivatives
Expression and purification of full-length, functionally active toxins has proven difficult in laboratories using alternative construct designs and expression systems. The ideal construct and expression system preferably do not segregate Clostridial toxins, because they contain coding sequences non-typical for the host; are not poisoned by entry of active toxin into the cytosol where it may disrupt the apparatus for regulated exocytosis, which is similar in most eukaryotes; and allow normal post-translational modification of the expressed toxins, particularly formation of disulfide bridges.
Two expression systems were tested for each toxin serotype A: baculovirus and E. coli. Because the baculovirus expression system was found to be most effective for expressing full-length BoNT A td, this was used as a starting point and benchmark for all the toxins. Though much concentration was centered on the baculovirus expression system, alternatives were evaluated, taking scale-up and cost into consideration, and work can be performed to optimize expression of all serotypes in these systems, as well as in other expression systems such as Pichia pastoris. 
Work that was performed to optimize expression and purification are described supra. The effect of these modifications on nativity of the toxin was evaluated in each case.
Prophetic Example 24
E. coli Expression System
Attempts to produce full-length BoNT A in E. coli resulted in a major C-terminally truncated propeptide which was significantly smaller than expected for the BoNT A propeptide (FIG. 4). In the future, the C-terminal composition of this product will be analyzed by microsequencing, identifying putative proteolytic cleavage sites specific to the E. coli system, and redesigning the pETcoco expression construct with amino acid substitutions designed to suppress this effect. It is possible the proteolysis site is similar to that recognized by trypsin, which has been demonstrated to cleave within the C-terminal BoNT A receptor-binding domain when applied in excessive amounts (Chaddock et al., “Expression and Purification of Catalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridium Botulinum Toxin Type A,” Protein Expr. Purif. 25:219-228 (2002), which is hereby incorporated by reference in its entirety). Expression of re-designed construct will use the advanced Rosetta-gami B (DE3) E. coli strain, as described infra.
Prophetic Example 25
Targeting Secretion to the Periplasm
Targeting recombinant proteins for secretion to the E. coli periplasm can improve stability and post-translational disulfide bond formation. The coding portion of the BoNT A td sequence will be subcloned into pET39b(+) vector (Novagen, Cat. #70090-3) which contains the signal required for export and periplasmic folding of target proteins. This system is designed for cloning and expression of peptide sequences fused with the 208 amino acids DsbA•TagTM. DsbA is a periplasmic enzyme that catalyzes the formation and isomerization of disulfide bonds (Rietsch et al., “An In vivo Pathway for Disulfide Bond Isomerization in Escherichia coli,” Proc. Natl. Acad. Sci. USA 93:13048-13053 (1996); Sone et al., “Differential In vivo Roles Played by DsbA and DsbC in the Formation of Protein Disulfide Bonds,” J. Biol. Chem. 272:10349-10352 (1997); Missiakas et al., “The Escherichia coli DsbC (xprA) Gene Encodes a Periplasmic Protein Involved in Disulfide Bond Formation,” EMBO J. 13:2013-2020 (1994); Zapun et al., “Structural and Functional Characterization of DsbC, a Protein Involved in Disulfide Bond Formation in Escherichia coli,” Biochemistry 34:5075-5089 (1995); Raina et al., “Making and Breaking Disulfide Bonds,” Annu. Rev. Microbiol. 51:179-202 (1997), which are hereby incorporated by reference in their entirety). It is possible that the degradation of BoNT A described infra occurs as a result of E. coli incompetence to properly form disulfide bridges for proteins which accumulate in the cytoplasm. The DsbA vector may enhance solubility and proper folding of recombinant BoNTs in the non-reducing periplasmic environment (Collins-Racie et al., “Production of Recombinant Bovine Enterokinase Catalytic Subunit in Escherichia coli Using the Novel Secretory Fusion Partner DsbA,” Biotechnology 13:982-987 (1995), which is hereby incorporated by reference in its entirety). Though the yield of recombinant proteins targeted to the periplasm is usually low, periplasmic expression in E. coli is worth continued consideration.
Prophetic Example 26
Pichia pastoris Expression System
Multi-copy Pichia pastoris expression kit (Invitrogen, Cat. #K1750-01) is used to obtain recombinant proteins. Recombinant plasmid on the backbone of the vector pPIC9K, carrying gene of interest and targeted for incorporation into Pichia genome is digested by restriction endonuclease SalI for linearization and transformed in the Pichia strains GS115 and KM71 by spheroplasting method with zymolyase, according to the supplied manufacturer's protocol. Primary and secondary rounds of the transformants selection on the histidine-deficient medium and in the presence of Geneticin is performed according to the same protocol. Protein expression is induced by the addition of methanol (0.5% final concentration) into the culture medium. Disrupted cells and medium are analyzed by SDS-PAGE and Western blotting.
Though the baculovirus expression system was found to provide satisfactory level of protein expression of BoNT A, recombinant protein expression in Pichia pastoris (methylotrophic yeast capable of metabolizing methanol as its sole carbon source) was evaluated because of the multiple reports describing successful expression in this system, including fragments of botulinum neurotoxin type A (Byrne et al., “Purification, Potency, and Efficacy of the botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate,” Infect. Immun. 66:4817-4822 (1998), which is hereby incorporated by reference in its entirety). This system has the advantages of low cost, post-translational modification of the recombinant proteins typical for eukaryotes, and low amounts of naturally secreted products, which facilitate purification of the recombinant proteins. The Pichia expression system also can provide better yields than the baculovirus system. Disadvantages of the Pichia system include cumbersome procedures of cloning into the Pichia genome and selection of multiple-copy recombinants, the possibility of extensive glycosylation of some recombinant proteins, and the possibility of premature termination of RNA transcripts synthesized from AT-rich templates, a known characteristic of the Clostridial toxin genes. These unwanted internal DNA features in BoNT genes were eliminated at the cloning stage. The system was the first to be tested with BoNT A td to establish benchmarks for comparison to other expression systems.
Prophetic Example 27
Engineering of the Expression Constructs Targeted for Secretion
The coding part of the modified N-terminally 6-His tagged BoNT A td was subcloned into vector pPIC9K, which provides the alpha-factor secretion signal from S. cerevisiae in the expression plasmid. This should result in secretion of the expressed protein into the medium. The construct was linearized by restriction endonuclease Sal I and transfected by spheroplasting method into KM71 and GS115 strains of Pichia pastoris. Primary selection of the transformants was performed by testing ability of the cells to grow on histidine-deficient media, trait deficient in the wild-type cells. Second round of selection was performed in the presence of antibiotic Geneticin which allowed the identification of clones with multiple inserts of the gene of interest by the correlation between the number of the copies of the gene of interest and increased concentration of Geneticin in the growth medium. The ability of identified clones to express protein of interest will be tested by growing cells in the methanol-containing medium.
Prophetic Example 28
Lengthening of the His Affinity Tag
The length of the histidine affinity tag at the N-termini of the BoNT A td were increased, and two more constructs—8-His and 12-His tagged were tested for their ability to confer higher affinity for Ni-NTA agarose in the recombinant BoNT products. This approach has been used successfully for other recombinant proteins which showed a similar decreased affinity for Ni-NTA affinity purification media (Ichtchenko et al., “Alpha-Latrotoxin Action Probed with Recombinant Toxin: Receptors Recruit Alpha-Latrotoxin but do not Transduce an Exocytotic Signal,” EMBO J. 17:6188-6199 (1998); Rudenko et al., “Structure of the LDL Receptor Extracellular Domain at Endosomal pH,” Science 298:2353-2358 (2002), which are hereby incorporated by reference in their entirety). If improved purification of recombinant BoNT A td can be achieved, at least two steps of ion-exchange chromatography can be omitted from the current purification scheme.
Example 29
Engineering the Non-Expression Plasmid pLitBoNTAME224A Containing the Full-Length Sequence of BoNT A ad
The plasmid encoding full-length BoNT A ad cDNA with protease-inactivating mutation E224>A was created by the site-directed mutagenesis of the plasmid pLitBoNTA with phosphorylated oligonucleotides









CBA18:



(SEQ ID NO: 46)



5′-pCCCGCGGTGACATTAGCACATGCACTTATACATGCTGG



and



 



CBA19:



(SEQ ID NO: 47)



5′-pCATGTGCTAATGTCACCGCGGGATCTGTAGCAAATTTG






using GeneTailorTM Site-Directed Mutagenesis System (Invitrogen, Cat. #12397-014) and Platinum® Pfx DNA Polymerase (Invitrogen, Cat. #11708-021), according to the protocol supplied by the manufacturer. The size of pLitBoNTAME224A is 6712 b.p. with 3900 b.p. coding sequence.

Example 30
Engineering of the Non-Expression Plasmid pLitGFPBoNTAHC, Containing Full-Length Sequence of BoNT A gfpd
The plasmid pLitGFPBoNTAHC, encoding chimeric protein where minimal essential catalytic domain of the BoNT A light chain was substituted with the GFP was created by the following protocol: 742 b.p. PCR product, obtained on plasmid pEGFP-N3 (Clontech, Cat. #632313) with oligonucleotides







CBA20:


(SEQ ID NO: 48)


5′-ATTAAGGATCCTGTGAGCAAGGGCGAGGAGCTGTTCACCG


and


 


CBA21:


(SEQ ID NO: 49)


5′-TATGAATTCAAACAATCCAGTAAAATTTTTCTTGTACAGCTCGTCCA


 


TGCC






and digested with restriction endocucleases BamHI and EcoRI and subcloned into pre-digested and dephosphorylated vector pLitBoNTALC, resulting in plasmid pLitGFPLC. 2615 b.p. DNA fragment from the vector pLitBoNTAHC, digested with restriction endonucleases XbaI and ApaI was subcloned into pre-digested and dephosphorylated vector pLitGFPLC, resulting in plasmid pLitGFPBoNTAHC. The size of pLitGFPBoNTAHC is 6216 b.p. with 3404 b.p. of coding sequence.

Example 31
Engineering of the Expression Plasmids pETCBoNTAME224A and pETCGFPBoNTAHC for the Expression of BoNT A ad and BoNT A gfpd in E. coli 
The plasmids were obtained by subcloning DNA fragments isolated from pLitmus-based vectors digested with NheI and NotI into pre-digested and dephosphorylated expression vector pETcoco2 (Novagen, Cat. #71148-3) and resulted in 16,194 b.p. BoNT A E224>A mutant expression vector pETCBoNTAME224A and 15,699 b.p. BoNT A chimeric vector pETCGFPBoNTAHC, where minimal essential catalytic domain of the BoNT A light chain was substituted with the GFP.
Example 32
Engineering of the Donor Plasmids pFBSecBoNTAME224A and pFBSecGFPBoNTAHC for the Expression of BoNT A ad and BoNT A gfpd in Baculovirus Expression System
The plasmids were obtained by the following protocol: 112 b.p. PCR product synthesized on plasmid pBac-3 (Novagen, Cat. #70088-3) with oligonucleotides







CBA 22:


(SEQ ID NO: 50)


5′-TAAGCGCGCAGAATTCTCTAGAATGCCCATGTTAAGCGCTATTG


and


 


CBA23:


(SEQ ID NO: 51)


5′-TAAGCTAGCGTGATGGTGGTGATGATGGACCATGGCC






and digested with restriction endonucleases BssHII and NheI was subcloned into pre-digested and dephosphorylated pLitmus-based vectors, resulting in plasmids pLitSecBoNTAME224A and pLitSecGFPBoNTAHC. DNA fragments from these vectors, obtained as a result of the digest with BssHII and NotI, were subcloned into pre-digested and dephosphorylated vector pFastBacTM1 (Invitrogen, Cat. #10360-014), resulting in 8764 b.p. pFBSecBoNTAME224A and 8568 b.p. pFBSecGFPBoNTAHC donor plasmids.

Example 33
Chimera Proteins that Target the Cytosol of Neurotoxin-Affected Neurons
A genetic engineering platform designed to produce BoNT antidotes that can effectively target the cytoplasm of BoNT-affected neurons has been developed. Antidotes designed pursuant to this platform have the potential to be effectively administered to a subject for extended time periods after exposure to toxic Clostridial neurotoxin, and would improve the practical logistics of administering antidote to large populations in an emergency setting. Using genetic constructs of the isolated BoNT A light chain, protein motifs are introduced to bind, inactivate, or otherwise mark toxic wild-type Clostridial neurotoxin (e.g., Clostridium botulinum) light chain for elimination from the cytosol of neurotoxin-affected neurons. Chimeric light chains with optimized antidote activity can subsequently be recombined with derivitized constructs of the Clostridial neurotoxin heavy chain to produce full-length Clostridial neurotoxin chimeras that can deliver antidote activity to the cytosol of Clostridial neurotoxin-affected neurons. Expression systems can be developed and tested (as described above) to ensure that the structural features and post-translational modifications responsible for native Clostridial neurotoxin trafficking are preserved. Produced Clostridial neurotoxin antidotes of this sort can effectively target neurotoxin-affected neurons when administered by oral or inhalational routes and can be used to rescue patients already experiencing symptoms of Clostridial neurotoxin intoxication (e.g., patients on an artificial respirator).
Using the plasmid encoding atoxic BoNT A light chain, a first library of BoNT A ad light chain chimeras containing SNARE motif peptides substituting light chain alpha-helix regions has been designed and created. The SNARE motif is recognized by all seven BoNT serotypes, and prior work has demonstrated physiological exocytosis (Rossetto et al., “SNARE Motif and Neurotoxins,” Nature 372:415-416 (1994), which is hereby incorporated by reference in its entirety). The chimeras are constructed to retain the interface responsible for BoNT light chain dimerization (Segelke et al., “Crystal Structure of Closridium Botulinum Neurotoxin Protease in a Product-Bound State: Evidence for Noncanonical Zinc Protease Activity,” Proc. Natl. Acad. Sci. USA 101:6888-6893 (2004), which is hereby incorporated by reference in its entirety), allowing them to preferentially bind to wild-type light chains and potentially inactivate or otherwise destabilize the toxic neurotoxin. Engineering of non-expression plasmids containing the full-length sequence of BoNT A (atoxic derivative, ad) with non-native SNARE motif peptides (illustrated in FIG. 11) to produce the light chain chimeric libraries is described in the following paragraphs.
Light Chain of BoNT A
The plasmid encoding mutated light chain of BoNT A cDNA with metalloprotease-inactivating mutation E224>A (pLitBoNTALCME224A) was created by the site-directed mutagenesis of the plasmid pLitBoNTALC with phosphorylated oligonucleotides







(SEQ ID NO: 46)


CBA18: 5′-pCCCGCGGTGACATTAGCACATGCACTTATACATGCTGG


and


 


(SEQ ID NO: 47)


CBA19: 5′-pCATGTGCTAATGTCACCGCGGGATCTGTAGCAAATTTG






using GeneTailorTM Site-Directed Mutagenesis System (Invitrogen, Cat. #12397-014) and Platinum® Pfx DNA Polymerase (Invitrogen, Cat. #11708-021), according to the protocol supplied by the manufacturer. The resulting plasmid pLitBoNTALCME224A is 4042 b.p. with a 1230 b.p. coding sequence.

Chimera 1
The non-expression plasmid pLitBoNTACH1, containing the full-length sequence of BoNT A ad with three SNARE motif peptides substituting BoNT A light chain alpha-helix 1, was created by site-directed mutagenesis of the plasmid pLitBoNTAME224A with phosphorylated oligonucleotides







CBCH1:


(SEQ ID NO: 52)


5′-pGAGTTGTTCGCCTTGCTCATCCAACATCTGCAACGCGTCAGCTCGG


 


TCATCCAACTCTGTACTTAAATATGTTGAATCATAATATGAAACTGG


and


 


CBCH2:


(SEQ ID NO: 53)


5′-pGAGCGCGAAATGGATGAAAACCTAGAGCAGGTGAGCGGCCGAGGAA


 


TACCATTTTGGGGTGGAAGTACAATAGATACAG






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit (Stratagene, Cat. #200502) with modification. The ExSiteTM DNA polymerase blend included in the kit was substituted with a blend consisting of 75% of TaKaRa LA Taq DNA polymerase (Takara, Cat. # RR002A) and 25% of Platinum® Pfx polymerase (Invitrogen, Cat. #11708-021). The mutagenesis reaction and selection of the mutant plasmid were performed according to the protocol, included in the original ExsiteTM PCR-Based Site-Directed Mutagenesis Kit. For selection purposes, two de novo endonuclease restriction sites—MluI and XhoI—were introduced into the plasmid.

Chimera 2
The non-expression plasmid pLitBoNTACH2, containing the full-length sequence of BoNT A ad with two SNARE motif peptides substituting BoNT A light chain alpha-helix 4, was created by site-directed mutagenesis of the plasmid pLitBoNTAME224A with phosphorylated oligonucleotides







CBCH3:


(SEQ ID NO: 54)


5′-pCGCGTCTGCCCTATCGTCTAGTTCATCTATAAACTTTGCATCATGT


 


CCCCC


and


 


CBCH4:


(SEQ ID NO: 55)


5′-pTTACAAATGCTAGACGAACAGGGAGAGCAGCTCGAGAGGCTTAATA


 


A AGCTAAATCAATAGTAGGTACTACTGC






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications, described above.

Chimera 3
The non-expression plasmid pLitBoNTACH3, containing the full-length sequence of BoNT A ad with five SNARE motifs peptides substituting BoNT A light chain alpha-helices 1 and 4, was created by site-directed mutagenesis of the plasmid pLitBoNTACH1 with phosphorylated oligonucleotides







CBCH3:


(SEQ ID NO: 54)


5′-pCGCGTCTGCCCTATCGTCTAGTTCATCTATAAACTTTGCATCATGT


 


CC CCC


and


 


CBCH4:


(SEQ ID NO: 55)


5′-pTTACAAATGCTAGACGAACAGGGAGAGCAGCTCGAGAGGC TTAAT


 


AAAGCTAAATCAATAGTAGGTACTACTGC






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications described above.

Chimera 4
The non-expression plasmid pLitBoNTACH4, containing the full-length sequence of BoNT A ad with three SNARE motif peptides substituting light chain alpha-helices 4 and 5, was created by site-directed mutagenesis of the plasmid pLitBoNTACH2 with phosphorylated oligonucleotides







CBCH5:


(SEQ ID NO: 56)


5′-pGCTTACTTGTTCCAAATTCTCGTCCATCTCTGAAGCAGTAGTACCT


 


AC TATTGATTTAGC


and


 


CBCH6:


(SEQ ID NO: 57)


5′-pGGCCGTCTCCTATCTGAAGATACATCTGG






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications described above. For the selection purposes, de novo endonuclease restriction site Eco52I was introduced into the plasmid.

Chimera 5
The non-expression plasmid pLitBoNTACH5, containing the full-length sequence of BoNT A ad with six SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, and 5, was created by site-directed mutagenesis of the plasmid pLitBoNTACH2 with phosphorylated oligonucleotides







CBCH5:


(SEQ ID NO: 56)


5′-pGCTTACTTGTTCCAAATTCTCGTCCATCTCTGAAGCAGTAGTACCT


 


AC TATTGATTTAGC


and


 


CBCH6:


(SEQ ID NO: 57)


5′-pGGCCGTCTCCTATCTGAAGATACATCTGG






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications described above. For the selection purposes, de novo endonuclease restriction site Eco52I was introduced into the plasmid.

Chimera 6
The non-expression plasmid pLitBoNTACH6, containing the full-length sequence of BoNT A ad with four SNARE motif peptides substituting BoNT A light chain alpha-helices 4, 5, and 6, was created by site-directed mutagenesis of the plasmid pLitBoNTACH4 with phosphorylated oligonucleotides









CBCH7:



(SEQ ID NO: 58)



5′-pAATTCATCCATGAAATCTACCGAAAATTTTCC



and



 



CBCH8:



(SEQ ID NO: 59)



5′-pCTTTGAACAGGTGGAGGAATTAACAGAGATTTACACAGAGG






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications described above. For the selection purposes, de novo endonuclease restriction site EcoRI was introduced into the plasmid.

Chimera 7
The non-expression plasmid pLitBoNTACH7, containing the full-length sequence of BoNT A ad with five SNARE motif peptides substituting BoNT A light chain alpha-helices 4, 5, 6, and 7, was created by site-directed mutagenesis of the plasmid pLitBoNTACH6 with phosphorylated oligonucleotides







CBCH9:


(SEQ ID NO: 60)


5′-pTCGAGCTCTGTGTAAATCTCTGTTAATTCC


and


 


CBCH10:


(SEQ ID NO: 61)


5′-pGGACATGCTGGAGAGTGGGAATCTTAACAGAAAAACATATTTGAAT


 


TTTG






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications described above. For the selection purposes, de novo endonuclease restriction site XhoI was introduced into the plasmid.

Chimera 8
The non-expression plasmid pLitBoNTACH8, containing the full-length sequence of BoNT A ad with seven SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, 5, and 6, was created by site-directed mutagenesis of the plasmid pLitBoNTACH5 with phosphorylated oligonucleotides









CBCH7:



(SEQ ID NO: 58)



5′-pAATTCATCCATGAAATCTACCGAAAATTTTCC



and



 



CBCH8:



(SEQ ID NO: 59)



5′-pCTTTGAACAGGTGGAGGAATTAACAGAGATTTACACAGAGG






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications described above. For the selection purposes, de novo endonuclease restriction site EcoRI was introduced into the plasmid.

Chimera 9
The non-expression plasmid pLitBoNTACH9, containing the full-length sequence of BoNT A ad with eight SNARE motif peptides substituting BoNT
A light chain alpha-helices 1, 4, 5, 6, and 7, was created by site-directed mutagenesis of the plasmid pLitBoNTACH8 with phosphorylated oligonucleotides







CBCH9:


(SEQ ID NO: 60)


5′-pTCGAGCTCTGTGTAAATCTCTGTTAATTCC


and


 


CBCH10:


(SEQ ID NO: 61)


5′-pGGACATGCTGGAGAGTGGGAATCTTAACAGAAAAACATATTTGAAT


 


TTTG






using ExsiteTM PCR-Based Site-Directed Mutagenesis Kit with the modifications described above. For the selection purposes, de novo endonuclease restriction site XhoI was introduced into the plasmid.

Green Fluorescence Protein
The non-expression plasmid pLitEGFP, containing the full-length sequence of GFP for the fusions with BoNT A light chain, BoNT A light chain ad, and BoNT A light chain ad chimeric derivatives, was created by subcloning  ̃750 b.p. product obtained by PCR on plasmid pEGFP-N3 (Clontech, Cat. #6080-1) with oligonucleotides







EGFPs:


(SEQ ID NO: 62)


5′-TATTACGCGTGCGCGCTATGAATTCTATAAGTTGCTAATGGTGAGCA


 


AGGGCGAGGAGCTGTTCACCGGG


and


 


EGFPa:


(SEQ ID NO: 63)


5′-ATTAGGGCCCCTATTACTTGTACAGCTCGTCCATGCCGAGAGTGATC


 


CC






and digested with restriction endonucleases MluI and ApaI into vector pLitmus38i (NEB, Cat. #N3538S) pre-digested with MluI and ApaI and dephosphorylated. The size of the resulting pLitEGFP was 3479 b.p.

Light Chain of BoNT A td Fused with EGFP
The non-expression vector pLitBoNTALCEGFP carrying light chain of BoNT A td, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the digest of the plasmid pLitBoNTALC with restriction endonucleases BssHII and EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of BoNT A ad Fused with EGFP
The non-expression vector pLitBoNTAME224ALCEGFP carrying the sequence of the light chain of BoNT A ad, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the digest of the plasmid pLitBoNTAME224A with restriction endonucleases BssHII and EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 1 Fused with EGFP
The non-expression vector pLitBoNTACH1EGFP, carrying the sequence of the BoNT A ad light chain with three SNARE motif peptides substituting BoNT A light chain alpha-helix 1, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the digest of the plasmid pLitBoNTACH1, with restriction endonucleases BssHII and EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 2 Fused with EGFP
The non-expression vector pLitBoNTACH2EGFP, carrying the sequence of the BoNT A ad light chain with two SNARE motif peptides substituting BoNT A light chain alpha-helix 4, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the digest of the plasmid pLitBoNTACH2 with restriction endonucleases BssHII and EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 3 Fused with EGFP
The non-expression vector pLitBoNTACH3EGFP, carrying the sequence of the BoNT A ad light chain with five SNARE motif peptides substituting BoNT A light chain alpha-helices 1 and 4, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the digest of the plasmid pLitBoNTACH3 with restriction endonucleases BssHII and EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 4 Fused with EGFP
The non-expression vector pLitBoNTACH4EGFP, carrying the sequence of the BoNT A ad light chain with three SNARE motif peptides substituting BoNT A light chain alpha-helices 4 and 5, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the digest of the plasmid pLitBoNTACH4 with restriction endonucleases BssHII and EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 5 Fused with EGFP
The non-expression vector pLitBoNTACHSEGFP, carrying the sequence of the BoNT A ad light chain with six SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, and 5, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment, obtained from the digest of the plasmid pLitBoNTACH5 with restriction endonucleases BssHII and EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimer 6 Fused with EGFP
The non-expression vector pLitBoNTACH6EGFP, carrying the sequence of the BoNT A ad light chain with four SNARE motif peptides substituting BoNT A light chain alpha-helices 4, 5, and 6, fused with EGFP, was created by subcloning 1296 b.p. DNA fragment, obtained from the incomplete digest with EcoRI of the 2019 b.p. DNA fragment, obtained from the plasmid pLitBoNTACH6, digested with restriction endonucleases BssHII and AlwNI, into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 7 Fused with EGFP
The non-expression vector pLitBoNTACH7EGFP, carrying the sequence of the BoNT A ad light chain with five SNARE motif peptides substituting BoNT A light chain alpha-helices 4, 5, 6, and 7, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment, obtained from the incomplete digest with EcoRI of the 2019 b.p. DNA fragment, obtained from the plasmid pLitBoNTACH7 digested with restriction endonucleases BssHII and AlwNI into vector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 8 Fused with EGFP
The non-expression vector pLitBoNTACH8EGFP, carrying the sequence of the BoNT A ad light chain with seven SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, 5, and 6, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the incomplete digest with EcoRI of the 1754 b.p. DNA fragment obtained from the plasmid pLitBoNTACH8 digested with restriction endonucleases BssHII and HincII into vector pLitEGFP pre-digested with restriction endonucleases BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Light Chain of Chimera 9 Fused with EGFP
The non-expression vector pLitBoNTACH9EGFP, carrying the sequence of the BoNT A ad light chain with eight SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, 5, 6, and 7, fused with EGFP, was created by subcloning the 1296 b.p. DNA fragment obtained from the incomplete digest with EcoRI of the 1754 b.p. DNA fragment obtained from the plasmid pLitBoNTACH9 digested with restriction endonucleases BssHII and HincII, into vector pLitEGFP pre-digested with restriction endonucleases BssHII and EcoRI and dephosphorylated. The size of the resulting plasmid was  ̃4800 b.p.
Sindbis Expression Vector—EGFP
The Sindbis expression vector pSinEGFP, carrying the EGFP sequence was created by subcloning the  ̃750 b.p. DNA fragment obtained from the plasmid pLitEGFP sequentially digested with restriction endonuclease EcoRI filled-in with Klenow fragment, and digested with restriction endonuclease ApaI into vector pSinRep5 (Invitrogen, Cat. #K750-1) pre-digested with restriction endonucleases StuI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃10,250 b.p.
Sindbis Expression Vector—BoNT A td Light Chain Fused with EGFP
The Sindbis expression vector pSinBoNTALCEGFP, carrying the sequence of BoNT A td light, chain fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTALCEGFP, digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—BoNT A ad Light Chain Fused with EGFP
The Sindbis expression vector pSinBoNTAME224ALCEGFP, carrying the sequence of BoNT A ad light, chain fused with EGFP was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTAME224ALCEGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 1 Fused with EGFP
The Sindbis expression vector pSinBoNTACH1EGFP, carrying the sequence of BoNT A ad light chain with three SNARE motif peptides substituting BoNT A light chain alpha-helix 1, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH1EGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 2 Fused with EGFP
The Sindbis expression vector pSinBoNTACH2EGFP, carrying the sequence of BoNT A ad light chain with two SNARE motif peptides substituting BoNT A light chain alpha-helix 4, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH2EGFP, digested with restriction endonucleases NheI and ApaI into vector pSinRep5 (Invitrogen, Cat. #K750-1) pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 3 Fused with EGFP
The Sindbis expression vector pSinBoNTACH3EGFP, carrying the sequence of BoNT A ad light chain with five SNARE motif peptides substituting BoNT A light chain alpha-helices 1 and 4, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH3EGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 4 Fused with EGFP
The Sindbis expression vector pSinBoNTACH4EGFP, carrying the sequence of BoNT A ad light chain with three SNARE motif peptides substituting BoNT A light chain alpha-helices 4 and 5, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH4EGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 5 Fused with EGFP
The Sindbis expression vector pSinBoNTACH5EGFP, carrying the sequence of BoNT A ad light chain with six SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, and 5, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACHSEGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 6 Fused with EGFP
The Sindbis expression vector pSinBoNTACH6EGFP, carrying the sequence of BoNT A ad light chain with four SNARE motif peptides substituting BoNT A light chain alpha-helices 4, 5, and 6, fused with EGFP, was created by subcloning  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH6EGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 7 Fused with EGFP
The Sindbis expression vector pSinBoNTACH7EGFP, carrying the sequence of BoNT A ad light chain with five SNARE motif peptides substituting BoNT A light chain alpha-helices 4, 5, 6, and 7, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH7EGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 8 Fused with EGFP
The Sindbis expression vector pSinBoNTACH8EGFP, carrying the sequence of BoNT A ad light chain with seven SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, 5, and 6, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH8EGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
Sindbis Expression Vector—Light Chain of Chimera 9 Fused with EGFP
The Sindbis expression vector pSinBoNTACH9EGFP, carrying the sequence of BoNT A ad light chain with eight SNARE motif peptides substituting BoNT A light chain alpha-helices 1, 4, 5, 6, and 7, fused with EGFP, was created by subcloning the  ̃2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH9EGFP digested with restriction endonucleases NheI and ApaI into vector pSinRep5 pre-digested with restriction endonucleases XbaI and ApaI and dephosphorylated. The size of the resulting plasmid was  ̃11,550 b.p.
The Sindbis expression vectors were prepared for RNA synthesis. The plasmids pSinEGFP, pSinBoNTALCEGFP, pSinBoNTAME224ALCEGFP, pSinBoNTACH1EGFP, pSinBoNTACH2EGFP, pSinBoNTACH3EGFP, pSinBoNTACH4EGFP, pSinBoNTACHSEGFP, pSinBoNTACH6EGFP, pSinBoNTACH7EGFP, pSinBoNTACH8EGFP, and pSinBoNTACH9EGFP were linearized by the digest with restriction endonuclease NotI. The linearized plasmids were used for the RNA synthesis according to the protocol supplied with Sindbis expression system kit (Invitrogen, Cat. #K750-1).
DSGXXS Motif Library
To further mark the antagonist wild-type BoNT A light chain complex for elimination, a second library of light chain chimeras will be produced. This library will incorporate the DSGXXS (SEQ ID NO: 64) motif into the chimeras produced in the first library. The motif DSGXXS is present in a variety of cytosolic proteins and has been shown to target them for degradation via the proteosome pathway upon its phosphorylation (Cardozo et al., “The SCF Ubiquitin Ligase: Insights Into a Molecular Machine,” Nat. Rev. Mol. Cell Biol. 5:739-751 (2004); Busino et al., “Degradation of Cdc25A by Beta-TrCP During S Phase and In Response to DNA Damage,” Nature 426:87-91 (2003), which are hereby incorporated by reference in their entirety). The motif will be positioned to cause minimal interference with the 3D structure of the ancestral protein (i.e., wild-type BoNT A light chain).
Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

## Claims

1. An isolated, physiologically active Clostridial neurotoxin comprising:
a light chain terminating at its C-terminus with a lysine residue of a highly specific protease cleavage site comprising three or more specific adjacent amino acid residues that are recognized by a highly specific protease to enable cleavage and
a heavy chain, wherein the light chain and the heavy chain are linked by a disulfide bond.

2. The isolated Clostridial neurotoxin according to claim 1, wherein the neurotoxin is from Clostridium botulinum.

3. The isolated Clostridial neurotoxin according to claim 2, wherein the Clostridium botulinum has a serotype selected from the group consisting of Clostridium botulinum serotype A, Clostridium botulinum serotype B, Clostridium botulinum serotype C, Clostridium botulinum serotype D, Clostridium botulinum serotype E, Clostridium botulinum serotype F, and Clostridium botulinum serotype G.

4. The isolated Clostridial neurotoxin according to claim 1, wherein said physiological activity is selected from the group consisting of toxin immunogenicity, trans- and intra-cellular trafficking, and cell recognition.

5. The isolated Clostridial neurotoxin according to claim 1, wherein the neurotoxin is toxic.

6. The isolated Clostridial neurotoxin according to claim 1, wherein the neurotoxin is atoxic.

7. The isolated Clostridial neurotoxin according to claim 6, wherein the neurotoxin has a disabling mutation in an active metalloprotease site.

8. The isolated Clostridial neurotoxin according to claim 6, wherein the light chain comprises a non-native motif capable of inactivating light chain metalloprotease activity in a toxic Clostridial neurotoxin.

9. The isolated Clostridial neurotoxin according to claim 1, wherein the neurotoxin further comprises:
a signal peptide coupled to the light chain, wherein the signal peptide is suitable to permit secretion of the neurotoxin from a eukaryotic cell to a medium and
an affinity tag located between the signal peptide and the light chain.

10. The isolated Clostridial neurotoxin according to claim 9, wherein the affinity tag is a hexahistidine affinity tag.

11. The isolated Clostridial neurotoxin according to claim 10, wherein the affinity tag comprises SEQ ID NO: 45.

12. The isolated Clostridial neurotoxin according to claim 1, wherein the heavy chain comprises a modification at a Lys amino acid residue adjacent to a receptor binding domain.

13. The isolated Clostridial neurotoxin according to claim 12, wherein the modification is a Lys to Asn substitution.

14. The isolated Clostridial neurotoxin according to claim 1, wherein the highly specific protease cleavage site is an enterokinase cleavage site of SEQ ID NO:24.

15. A vaccine or antidote comprising the isolated Clostridial neurotoxin according to claim 6.

16. The vaccine or antidote according to claim 15, wherein the neurotoxin is from Clostridium botulinum.

17. The vaccine or antidote according to claim 16, wherein the Clostridium botulinum has a serotype selected from the group consisting of Clostridium botulinum serotype A, Clostridium botulinum serotype B, Clostridium botulinum serotype C, Clostridium botulinum serotype D, Clostridium botulinum serotype E, Clostridium botulinum serotype F, and Clostridium botulinum serotype G.

18. The vaccine or antidote according to claim 15, wherein the neurotoxin has a disabling mutation in an active metalloprotease site.

19. The vaccine or antidote according to claim 15, wherein said physiological activity is selected from the group consisting of toxin immunogenicity, trans- and intra-cellular trafficking, and cell recognition.

20. A method of immunizing a subject against toxic effects of a Clostridial neurotoxin, said method comprising:
administering the vaccine according to claim 15 to the subject under conditions effective to immunize the subject against toxic effects of Clostridial neurotoxin.

21. The method according to claim 20, wherein the neurotoxin is from Clostridium botulinum.

22. The method according to claim 21, wherein the Clostridium botulinum has a serotype selected from the group consisting of Clostridium botulinum serotype A, Clostridium botulinum serotype B, Clostridium botulinum serotype C, Clostridium botulinum serotype D, Clostridium botulinum serotype E, Clostridium botulinum serotype F, and Clostridium botulinum serotype G.

23. The method according to claim 20, wherein the neurotoxin has a disabling mutation in an active metalloprotease site.

24. The method according to claim 20 further comprising:
administering a booster of the vaccine to the subject under conditions effective to enhance immunization of the subject.

25. A method of treating a subject for toxic effects of a Clostridial neurotoxin, said method comprising:
administering the antidote according to claim 15 to the subject under conditions effective to treat the subject for toxic effects of Clostridial neurotoxin.

26. The method according to claim 25, wherein the neurotoxin is from Clostridium botulinum.

27. The method according to claim 26, wherein the Clostridium botulinum has a serotype selected from the group consisting of Clostridium botulinum serotype A, Clostridium botulinum serotype B, Clostridium botulinum serotype C, Clostridium botulinum serotype D, Clostridium botulinum serotype E, Clostridium botulinum serotype F, and Clostridium botulinum serotype G.

28. The method according to claim 25, wherein the neurotoxin has a disabling mutation in an active metalloprotease site.

29. A treatment method comprising:
contacting a patient with an isolated Clostridial neurotoxin according to claim 5 under conditions effective to treat the patient.