US 200901 17157A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0117157 A1 Brin et al. (43) Pub. Date: May 7, 2009
(54) METHODS OF TREATING (22) Filed: Oct. 21, 2008 UROGENTAL-NEUROLOGICAL O O DSORDERS USING MODIFIED Related U.S. Application Data CLOSTRIDAL TOXINS (60) Provisional application No. 60/982,021, filed on Oct. 23, 2007, provisional application No. 61/076,228, (75) Inventors: Mitchell F. Brin, Newport Beach, filed on Jun. 27, 2008. CA (US); Joseph Francis, Aliso s Viejo, CA (US); Kei Roger Aoki, Publication Classification Coto de Caza, CA (US) (51) Int. Cl Correspondence Address: A6139/08 (2006.01) ALLERGAN, INC. (52) U.S. Cl...... 424/239.1; 424/190.1 2525 DUPONT DRIVE, T2-7H (57) ABSTRACT IRVINE, CA 92612-1599 (US) The present specification discloses modified Clostridial tox (73) Assignee: Allergan, Inc. ins, compositions comprising Such toxins and methods of treating urogenital-neurological disorders in a mammal using (21) Appl. No.: 12/255,369 Such modified Clostridial toxins and compositions. Patent Application Publication May 7, 2009 Sheet 1 of 7 US 2009/01171.57 A1
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METHODS OF TREATING of Clostridial toxin therapies beyond its current myo-relaxant UROGENTAL-NEUROLOGICAL applications to treat sensory nerve-based ailment, such as, DSORDERS USING MODIFIED e.g., various kinds of chronic pain, neurogenic inflammation CLOSTRIDAL TOXINS and urogenital disorders, as well as other disorders, such as, e.g., pancreatitis. One approach that is currently being exploited to expand Clostridial toxin-based therapies 0001. This patent application claims priority pursuant to involves modifying a Clostridial toxin so that the modified 35 U.S.C. S 119(e) to U.S. Provisional Patent Application Ser. toxin has an altered cell targeting capability for a non No. 60/982,021 filed Oct. 23, 2007, and U.S. Provisional Clostridial toxin target cell. This re-targeted capability is Patent Application Ser. No. 61/076.228 filed Jun. 27, 2008, achieved by replacing a naturally-occurring targeting domain each of which is hereby incorporated by reference in its of a Clostridial toxin with a targeting domain showing a entirety. selective binding activity for a receptor present on a non 0002 The ability of Clostridial toxins, such as, e.g., Botu Clostridial toxin target cell. Such modifications to a targeting linum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, domain result in a modified toxin that is able to selectively BoNT/D, BoNT/E, BoNT/F and BoNT/G, and Tetanus neu bind to a non-Clostridial toxin receptor (target receptor) rotoxin (TeNT), to inhibit neuronal transmission are being exploited in a wide variety of therapeutic and cosmetic appli present on a non-Clostridial toxin target cell (re-targeted). A cations, see e.g., William J. Lipham, COSMETIC AND CLINICAL modified Clostridial toxin with a targeting activity for a non APPLICATIONS OF BOTULINUM TOXIN (Slack, Inc., 2004). Clostridial toxin target cell can bind to a receptor present on Clostridial toxins commercially available as pharmaceutical the non-Clostridial toxin target cell, translocate into the cyto compositions include, BoNT/A preparations, such as, e.g., plasm, and exert its proteolytic effect on the SNARE complex BOTOX(R) (Allergan, Inc., Irvine, Calif.), DysportR/Re of the non-Clostridial toxin target cell. loxin R. (Beaufour Ipsen, Porton Down, England), Linurase(R) 0005. The present specification discloses modified (Prollenium, Inc., Ontario, Canada), NeuronoXR (Medy-Tox, Clostridial toxin compositions and methods for treating an Inc., Ochang-myeon, South Korea) BTX-A (Lanzhou Insti individual Suffering from a nociceptive sensory neuron-me tute Biological Products, China) and Xeomin R (Merz Phar diated urogenital disorder. This is accomplished by adminis maceuticals, GmbH., Frankfurt, Germany); and BoNT/B tering a therapeutically effective amount of a composition preparations, such as, e.g., MyoBlocTM/NeuroBlocTM (Elan comprising a modified Clostridial toxin to an individual in Pharmaceuticals, San Francisco, Calif.). As an example, need thereof. The disclosed methods provide a safe, inexpen BOTOX(R) is currently approved in one or more countries for sive, out patient-based treatment for the treatment of urogeni the following indications: achalasia, adult spasticity, anal fis tal-neurological disorders. Sure, back pain, blepharospasm, bruxism, cervical dystonia, 0006 Thus, aspects of the present invention provide a essential tremor, glabellar lines or hyperkinetic facial lines, composition comprising a modified Clostridial toxin com headache, hemifacial spasm, hyperactivity of bladder, hyper prising an opioid peptide binding domain, a Clostridial toxin hidrosis, juvenile cerebral palsy, multiple Sclerosis, myo translocation domain and a Clostridial toxin enzymatic clonic disorders, nasal labial lines, spasmodic dysphonia, domain. Modified Clostridial toxins useful for the develop strabismus and VII nerve disorder. ment of Such compositions are described in, e.g., Steward, L. 0003 Clostridial toxin therapies are successfully used for E. et al., Modified Clostridial Toxins with Enhanced Trans many indications. Generally, administration of a Clostridial location Capabilities and Altered Targeting Activity For Non toxin treatment is well tolerated. However, toxin administra Clostridial Toxin Target Cells, U.S. patent application Ser. tion in some applications can be challenging because of the No. 1 1/776,075 (Jul 11, 2007); Dolly, J. O. et al., Activatable larger doses required to achieve a beneficial effect. Larger Clostridial Toxins, U.S. patent application Ser. No. 1 1/829, doses can increase the likelihood that the toxin may move 475 (Jul 27, 2007); Foster, K. A. et al., Fusion Proteins, through the interstitial fluids and the circulatory systems, International Patent Publication WO 2006/059093 (Jun. 8, Such as, e.g., the cardiovascular system and the lymphatic 2006); and Foster, K. A. et al., Non-Cytotoxic Protein Con system, of the body, resulting in the undesirable dispersal of jugates, International Patent Publication WO 2006/059105 the toxin to areas not targeted for toxin treatment. Such dis (Jun. 8, 2006), each of which is incorporated by reference in persal can lead to undesirable side effects, such as, e.g., inhi its entirety. A composition comprising a modified Clostridial bition of neurotransmitter release in neurons not targeted for toxin can be a pharmaceutical composition. Such a pharma treatment or paralysis of a muscle not targeted for treatment. ceutical composition can comprise, in addition to a modified For example, a patient administered a therapeutically effec Clostridial toxin, a pharmaceutical carrier, a pharmaceutical tive amount of a BoNT/Atreatment into the neck muscles for component, or both. torticollis may develop dysphagia because of dispersal of the 0007. Other aspects of the present invention provide a toxin into the oropharynx. As another example, a patient method of treating urogenital-neurological disorder in a administered a therapeutically effective amount of a BoNT/A mammal, the method comprising the step of administering to treatment into the bladder for overactive bladder may develop the mammal atherapeutically effective amount of a compo dry month and/or dry eyes. Thus, there remains a need for sition including a modified Clostridial toxin comprising an improved Clostridial toxins that are effective at the site of opioid peptide binding domain, a Clostridial toxin transloca treatment, but have negligible to minimal effects in areas not tion domain and a Clostridial toxin enzymatic domain. It is targeted for a toxin treatment. envisioned that any modified Clostridial toxin disclosed in the 0004 A Clostridial toxin treatment inhibits neurotrans present specification can be used, including those disclosed mitter release by disrupting the exocytotic process used to in, e.g., Steward, supra, (2007); Dolly, supra, (2007); Foster, secret the neurotransmitter into the synaptic cleft. There is a supra, WO 2006/059093 (2006); and Foster, supra, WO 2006/ great desire by the pharmaceutical industry to expand the use 059105 (Jun. 8, 2006). US 2009/01171.57 A1 May 7, 2009
0008. Other aspects of the present invention provide a use peptide binding domain. The di-chain loop region located of a modified Clostridial toxin comprising an opioid peptide between the translocation and enzymatic domains is depicted binding domain, a Clostridial toxin translocation domain and by the double SS bracket. This region comprises an endog a Clostridial toxin enzymatic domain in the manufacturing a enous di-chain loop protease cleavage site that upon pro medicament for treating urogenital-neurological disorderina teolytic cleavage with a naturally-occurring protease, such as, mammal, the use comprising the step of administering to the e.g., an endogenous Clostridial toxin protease or a naturally mammal atherapeutically effective amount of a composition occurring protease produced in the environment, converts the including a modified Clostridial toxin comprising an opioid single-chain form of the toxin into the di-chain form. Above peptide binding domain, a Clostridial toxin translocation the single-chain form, the HCC region of the Clostridial toxin domain and a Clostridial toxin enzymatic domain. It is envi binding domain is depicted. This region comprises the B-tre sioned that any modified Clostridial toxin disclosed in the foil domain which comprises in a amino to carboxyl linear present specification can be used, including those disclosed organization an O-fold, a B4/B5 hairpin turn, a B-fold, a B8/B9 in, e.g., Steward, supra, (2007); Dolly, supra, (2007); Foster, hairpin turn and a Y-fold. supra, WO 2006/059093 (2006); and Foster, supra, WO 2006/ 0012 FIG. 3 shows modified Clostridial toxins with an 059105 (Jun. 8, 2006). enhanced targeting domain located at the amino terminus of 0009. Other aspects of the present invention provide a the modified toxin. FIG.3A depicts the single-chain polypep modified Clostridial toxin comprising an opioid peptide bind tide form of a modified Clostridial toxin with an amino to ing domain, a Clostridial toxin translocation domain and a carboxyl linear organization comprising a binding element, a Clostridial toxin enzymatic domain for treating urogenital translocation element, a di-chain loop region comprising an neurological disorder in a mammal, the use comprising the exogenous protease cleavage site (P), and a therapeutic ele step of administering to the mammal atherapeutically effec ment. Upon proteolytic cleavage with a Pprotease, the single tive amount of a composition including a modified Clostridial chainform of the toxin is converted to the di-chainform. FIG. toxin comprising an opioid peptide binding domain, a 3B depicts the single polypeptide form of a modified Clostridial toxin translocation domain and a Clostridial toxin Clostridial toxin with an amino to carboxyl linear organiza enzymatic domain. It is envisioned that any modified tion comprising a binding element, a therapeutic element, a Clostridial toxin disclosed in the present specification can be di-chain loop region comprising an exogenous protease used, including those disclosed in, e.g., Steward, Supra, cleavage site (P), and a translocation element. Upon pro (2007); Dolly, supra, (2007); Foster, supra, WO 2006/059093 teolytic cleavage with a P protease, the single-chain form of (2006); and Foster, supra, WO 2006/059105 (Jun. 8, 2006). the toxin is converted to the di-chain form. 0013 FIG. 4 shows modified Clostridial toxins with an BRIEF DESCRIPTION OF THE DRAWINGS enhanced targeting domain located between the other two 0010 FIG. 1 shows a schematic of the current paradigm of domains. FIG. 4A depicts the single polypeptide form of a neurotransmitter release and Clostridial toxin intoxication in modified Clostridial toxin with an amino to carboxyl linear a central and peripheral neuron. FIG. 1A shows a schematic organization comprising a therapeutic element, a di-chain for the neurotransmitter release mechanism of a central and loop region comprising an exogenous protease cleavage site peripheral neuron. The release process can be described as (P), a binding element, and a translocation element. Upon comprising two steps: 1) vesicle docking, where the vesicle proteolytic cleavage with a P protease, the single-chain form bound SNARE protein of a vesicle containing neurotransmit of the toxin is converted to the di-chain form. FIG. 4B depicts ter molecules associates with the membrane-bound SNARE the single polypeptide form of a modified Clostridial toxin proteins located at the plasma membrane; and 2) neurotrans with an amino to carboxyl linear organization comprising a mitter release, where the vesicle fuses with the plasma mem translocation element, a di-chain loop region comprising an brane and the neurotransmitter molecules are exocytosed. exogenous protease cleavage site (P), a binding element, and FIG.1B shows a schematic of the intoxication mechanism for a therapeutic element. Upon proteolytic cleavage with a P tetanus and botulinum toxin activity in a central and periph protease, the single-chain form of the toxin is converted to the eral neuron. This intoxication process can be described as di-chain form. FIG. 4C depicts the single polypeptide form of comprising four steps: 1) receptor binding, where a a modified Clostridial toxin with an amino to carboxyl linear Clostridial toxin binds to a Clostridial receptor system and organization comprising atherapeutic element, a binding ele initiates the intoxication process; 2) complex internalization, ment, a di-chain loop region comprising an exogenous pro where after toxin binding, a vesicle containing the toxin/ tease cleavage site (P), and a translocation element. Upon receptor System complex is endocytosed into the cell; 3) light proteolytic cleavage with a P protease, the single-chain form chain translocation, where multiple events are thought to of the toxin is converted to the di-chain form. FIG. 4D depicts occur, including, e.g., changes in the internal pH of the the single polypeptide form of a modified Clostridial toxin vesicle, formation of a channel pore comprising the HN with an amino to carboxyl linear organization comprising a domain of the Clostridial toxin heavy chain, separation of the translocation element, a binding element, a di-chain loop Clostridial toxin light chain from the heavy chain, and release region comprising an exogenous protease cleavage site (P), of the active light chain and 4) enzymatic target modification, and a therapeutic element. Upon proteolytic cleavage with a P where the activate light chain of Clostridial toxin proteolyti protease, the single-chain form of the toxin is converted to the cally cleaves its target SNARE substrate, such as, e.g., SNAP di-chain form. 25, VAMP or Syntaxin, thereby preventing vesicle docking 0014 FIG. 5 shows modified Clostridial toxins with an and neurotransmitter release. enhanced targeting domain located at the carboxyl terminus 0011 FIG. 2 shows the domain organization of naturally of the modified toxin. FIG. 5A depicts the single polypeptide occurring Clostridial toxins. The single-chain form depicts form of a modified Clostridial toxin with an amino to car the amino to carboxyl linear organization comprising an boxyl linear organization comprising atherapeutic element, a enzymatic domain, a translocation domain, and an opioid di-chain loop region comprising an exogenous protease US 2009/01171.57 A1 May 7, 2009
cleavage site (P), a translocation element, and a binding ele ment. Upon proteolytic cleavage with a Pprotease, the single TABLE 1 chain form of the toxin is converted to the di-chainform. FIG. 5B depicts the single polypeptide form of a modified Clostridial Toxin Reference Sequences and Regions Clostridial toxin with an amino to carboxyl linear organiza Toxin SEQ ID NO: LC Hy H. tion comprising a translocation element, a di-chain loop BONTA 1 M1-K448 A449-K871 N872-L1296 region comprising an exogenous protease cleavage site (P), a BONTB 2 M1-K441 A442-S858 E859-E1291 therapeutic element, and a binding element. Upon proteolytic BONT, C1 3 M1-K449 T450-N866 N867-E1291 cleavage with a P protease, the single-chain form of the toxin BONTD 4 M1-R445 D446-N862 S863-E1276 BONTE 5 M1-R422 K423-K845 R846-K1252 is converted to the di-chain form. BONTF 6 M1-K439 A440-K864 K865-E1274 00.15 Aspects of the present invention provide, in part, a BONTAG 7 M1-K446 S447-S863 N864-E1297 TeNT 8 M1-A457 S458-V879 I880-D1315 modified Clostridial toxin. As used herein, a “modified BaNT 9 M1-K431 N432-857 I858-E 1268 Clostridial toxin' means any molecule comprising an opioid BNT 10 M1-R422 K423-I847 Y1O86-K1251 peptide binding domain, a Clostridial toxin translocation domain and a Clostridial toxin enzymatic domain. Exemplary 0018. The binding, translocation and enzymatic activity of modified Clostridial toxins useful to practice aspects of the these three functional domains are all necessary for toxicity. present invention are disclosed in, e.g., Steward, Supra, While all details of this process are not yet precisely known, (2007); Dolly, supra, (2007); Foster, supra, WO 2006/059093 the overall cellular intoxication mechanism whereby (2006); Foster, supra, WO 2006/059105 (Jun. 8, 2006). Clostridial toxins enter a neuron and inhibit neurotransmitter 0016 Clostridia toxins produced by Clostridium botuli release is similar, regardless of serotype or Subtype. Although the applicants have no wish to be limited by the following num, Clostridium tetani, Clostridium baratii and Clostridium description, the intoxication mechanism can be described as butyricum are the most widely used in therapeutic and cos comprising at least four steps: 1) receptor binding, 2) com metic treatments of humans and other mammals. Strains of C. plex internalization, 3) light chain translocation, and 4) enzy botulinum produce seven antigenically-distinct types of matic target modification (see FIG.1). The process is initiated Botulinum toxins (BoNTs), which have been identified by when the H domain of a Clostridial toxin binds to a toxin investigating botulism outbreaks in man (BoNT/A, 7B, /E and specific receptor system located on the plasma membrane /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/ Surface of a target cell. The binding specificity of a receptor G). BoNTs possess approximately 35% amino acid identity complex is thought to be achieved, in part, by specific com with each other and share the same functional domain orga binations of gangliosides and protein receptors that appear to nization and overall structural architecture. It is recognized distinctly comprise each Clostridial toxin receptor complex. by those of skill in the art that within each type of Clostridial Once bound, the toxin/receptor complexes are internalized by toxin there can be subtypes that differ somewhat in their endocytosis and the internalized vesicles are sorted to specific intracellular routes. The translocation step appears to be trig amino acid sequence, and also in the nucleic acids encoding gered by the acidification of the vesicle compartment. This these proteins. For example, there are presently four BoNT/A process seems to initiate two important pH-dependent struc subtypes, BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4, tural rearrangements that increase hydrophobicity and pro with specific Subtypes showing approximately 89% amino mote formation di-chain form of the toxin. Once activated, acid identity when compared to another BoNT/A subtype. light chain endopeptidase of the toxin is released from the While all seven BoNT serotypes have similar structure and intracellular vesicle into the cytosol where it appears to spe pharmacological properties, each also displays heteroge cifically targets one of three known core components of the neous bacteriological characteristics. In contrast, tetanus neurotransmitter release apparatus. These core proteins, toxin (TeNT) is produced by a uniform group of C. tetani. vesicle-associated membrane protein (VAMP)/synaptobre Two other species of Clostridia, C. baratii and C. butyricum, vin, synaptosomal-associated protein of 25 kDa (SNAP-25) also produce toxins, BaNT and BuNT respectively, which are and Syntaxin, are necessary for synaptic vesicle docking and similar to BoNT/F and BoNT/E, respectively. fusion at the nerve terminal and constitute members of the 0017. Each mature di-chain molecule comprises three soluble N-ethylmaleimide-sensitive factor-attachment pro functionally distinct domains: 1) an enzymatic domain tein-receptor (SNARE) family. BoNT/A and BoNT/E cleave located in the LC that includes a metalloprotease region con SNAP-25 in the carboxyl-terminal region, releasing a nine or taining a zinc-dependent endopeptidase activity which spe twenty-six amino acid segment, respectively, and BoNT/C1 cifically targets core components of the neurotransmitter also cleaves SNAP-25 near the carboxyl-terminus. The botu linum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, release apparatus; 2) a translocation domain contained within and tetanus toxin, act on the conserved central portion of the amino-terminal half of the HC(H) that facilitates release VAMP, and release the amino-terminal portion of VAMP into of the LC from intracellular vesicles into the cytoplasm of the the cytosol. BoNT/C1 cleaves syntaxinata single site near the target cell; and 3) a binding domain found within the car cytosolic membrane Surface. The selective proteolysis of syn boxyl-terminal half of the HC (H) that determines the bind aptic SNAREs accounts for the block of neurotransmitter ing activity and binding specificity of the toxinto the receptor release caused by Clostridial toxins in vivo. The SNARE complex located at the surface of the target cell. The H, protein targets of Clostridial toxins are common to exocytosis domain comprises two distinct structural features of roughly in a variety of non-neuronal types; in these cells, as in neu equal size that indicate function and are designated the Hy rons, light chain peptidase activity inhibits exocytosis, see, and H, subdomains. Table 1 gives approximate boundary e.g., Yann Humeau et al. How Botulinum and Tetanus Neu regions for each domain found in exemplary Clostridial tox rotoxins Block Neurotransmitter Release, 82(5) Biochimie. 1S. 427-446 (2000); Kathryn Turton et al., Botulinum and Teta US 2009/01171.57 A1 May 7, 2009
nus Neurotoxins. Structure, Function and Therapeutic Util variant comprising amino acids 1-445 of SEQID NO. 4 will ity, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna have at least one amino acid difference, such as, e.g., an amino Lalli et al., The Journey of Tetanus and Botulinum Neurotox acid Substitution, deletion or addition, as compared to the ins in Neurons, 11 (9) Trends Microbiol. 431-437, (2003). amino acid region 1-445 of SEQID NO: 4; a BoNT/E enzy 0019. In an aspect of the invention, a modified Clostridial matic domain variant comprising amino acids 1-422 of SEQ toxin comprises, in part, a Clostridial toxin enzymatic ID NO: 5 will have at least one amino acid difference, such as, domain. As used herein, the term “Clostridial toxin enzymatic e.g., an amino acid Substitution, deletion or addition, as com domain” means any Clostridial toxin polypeptide that can pared to the amino acid region 1-422 of SEQ ID NO: 5; a execute the enzymatic target modification step of the intoxi BoNT/F enzymatic domain variant comprising amino acids cation process. Thus, a Clostridial toxin enzymatic domain 1-439 of SEQ ID NO: 6 will have at least one amino acid specifically targets a Clostridial toxin Substrate and encom difference. Such as, e.g., an amino acid Substitution, deletion passes the proteolytic cleavage of a Clostridial toxin Sub or addition, as compared to the amino acid region 1-439 of strate, such as, e.g., SNARE proteins like a SNAP-25 sub SEQ ID NO: 6; a BoNT/G enzymatic domain variant com strate, a VAMP substrate and a Syntaxin substrate. Non prising amino acids 1-446 of SEQID NO: 7 will have at least limiting examples of a Clostridial toxin enzymatic domain one amino acid difference, such as, e.g., an amino acid Sub include, e.g., a BoNT/A enzymatic domain, a BoNT/B enzy stitution, deletion or addition, as compared to the amino acid matic domain, a BoNT/C1 enzymatic domain, a BoNT/D region 1-446 of SEQ ID NO: 7; and a TeNT enzymatic enzymatic domain, a BoNT/E enzymatic domain, a BoNT/F domain variant comprising amino acids 1-457 of SEQID NO: enzymatic domain, a BoNT/G enzymatic domain, a TeNT 8 will have at least one amino acid difference, Such as, e.g., an enzymatic domain, a BaNT enzymatic domain, and a BuNT amino acid Substitution, deletion or addition, as compared to enzymatic domain. Other non-limiting examples of a the amino acid region 1-457 of SEQID NO: 8. Clostridial toxin enzymatic domain include, e.g., amino acids 0022. It is recognized by those of skill in the art that within 1-448 of SEQID NO: 1, amino acids 1-441 of SEQID NO: 2, each serotype of Clostridial toxin there can be naturally amino acids 1-449 of SEQID NO:3, amino acids 1-445 of occurring Clostridial toxin enzymatic domain variants that SEQ ID NO: 4, amino acids 1-422 of SEQID NO: 5, amino differ somewhat in their amino acid sequence, and also in the acids 1-439 of SEQID NO: 6, amino acids 1-446 of SEQ ID nucleic acids encoding these proteins. For example, there are NO: 7, amino acids 1-457 of SEQ ID NO: 8, amino acids presently five BoNT/A subtypes, BoNT/A1, BoNT/A2, 1-431 of SEQID NO: 9, and amino acids 1-422 of SEQ ID BoNT/A3, BoNT/A4, and BoNT/A5, with specific enzy NO: 1.O. matic domain subtypes showing approximately 95% amino 0020 A Clostridial toxin enzymatic domain includes, acid identity when compared to another BoNT/A enzymatic without limitation, naturally occurring Clostridial toxin enzy domain Subtype. As used herein, the term “naturally occur matic domain variants, such as, e.g., Clostridial toxin enzy ring Clostridial toxin enzymatic domain variant’ means any matic domain isoforms and Clostridial toxin enzymatic Clostridial toxin enzymatic domain produced by a naturally domain subtypes; non-naturally occurring Clostridial toxin occurring process, including, without limitation, Clostridial enzymatic domain variants, such as, e.g., conservative toxin enzymatic domain isoforms produced from alterna Clostridial toxin enzymatic domain variants, non-conserva tively-spliced transcripts, Clostridial toxin enzymatic domain tive Clostridial toxin enzymatic domain variants, Clostridial isoforms produced by spontaneous mutation and Clostridial toxin enzymatic domain chimerics, active Clostridial toxin toxin enzymatic domain Subtypes. A naturally occurring enzymatic domain fragments thereof, or any combination Clostridial toxin enzymatic domain variant can function in thereof. substantially the same manner as the reference Clostridial 0021. As used herein, the term “Clostridial toxin enzy toxin enzymatic domain on which the naturally occurring matic domain variant, whether naturally-occurring or non Clostridial toxin enzymatic domain variant is based, and can naturally-occurring, means a Clostridial toxin enzymatic be substituted for the reference Clostridial toxin enzymatic domain that has at least one amino acid change from the domain in any aspect of the present invention. A naturally corresponding region of the disclosed reference sequences occurring Clostridial toxin enzymatic domain variant may (Table 1) and can be described in percent identity to the Substitute one or more amino acids, two or more amino acids, corresponding region of that reference sequence. Unless three or more amino acids, four or more amino acids, five or expressly indicated, Clostridial toxin enzymatic domain vari more amino acids, ten or more amino acids, 20 or more amino ants useful to practice disclosed embodiments are variants acids, 30 or more amino acids, 40 or more amino acids, 50 or that execute the enzymatic target modification step of the more amino acids or 100 or more amino acids from the intoxication process. As non-limiting examples, a BoNT/A reference Clostridial toxin enzymatic domain on which the enzymatic domain variant comprising amino acids 1-448 of naturally occurring Clostridial toxin enzymatic domain vari SEQID NO: 1 will have at least one amino acid difference, ant is based. A naturally occurring Clostridial toxin enzy Such as, e.g., an amino acid Substitution, deletion or addition, matic domain variant can also Substitute at least 10 contigu as compared to the amino acid region 1-448 of SEQID NO: ous amino acids, at least 15 contiguous amino acids, at least 1; a BoNT/B enzymatic domain variant comprising amino 20 contiguous amino acids, or at least 25 contiguous amino acids 1-441 of SEQID NO: 2 will have at least one amino acid acids from the reference Clostridial toxin enzymatic domain difference. Such as, e.g., an amino acid Substitution, deletion on which the naturally occurring Clostridial toxin enzymatic or addition, as compared to the amino acid region 1-441 of domain variant is based, that possess at least 50% amino acid SEQID NO: 2; a BoNT/C1 enzymatic domain variant com identity, 65% amino acid identity, 75% amino acid identity, prising amino acids 1-449 of SEQID NO:3 will have at least 85% amino acid identity or 95% amino acid identity to the one amino acid difference, such as, e.g., an amino acid Sub reference Clostridial toxin enzymatic domain on which the stitution, deletion or addition, as compared to the amino acid naturally occurring Clostridial toxin enzymatic domain vari region 1-449 of SEQID NO:3: a BoNT/Denzymatic domain ant is based. US 2009/01171.57 A1 May 7, 2009
0023. A non-limiting examples of a naturally occurring acids, 40 or more amino acids, 50 or more amino acids, 100 or Clostridial toxin enzymatic domain variant is a Clostridial more amino acids, or 200 or more amino acids, from the toxin enzymatic domain isoform Such as, e.g., a BoNT/A reference Clostridial toxin enzymatic domain on which the enzymatic domain isoform, a BoNT/B enzymatic domain conservative Clostridial toxin enzymatic domain variant is isoform, a BoNT/C1 enzymatic domain isoform, a BoNT/D based. A conservative Clostridial toxin enzymatic domain enzymatic domain isoform, a BoNT/E enzymatic domain variant can also substitute at least 10 contiguous amino acids, isoform, a BoNT/F enzymatic domain isoform, a BoNT/G at least 15 contiguous amino acids, at least 20 contiguous enzymatic domain isoform, and a TeNT enzymatic domain amino acids, or at least 25 contiguous amino acids from the isoform. A Clostridial toxin enzymatic domain isoform can reference Clostridial toxin enzymatic domain on which the function in Substantially the same manner as the reference conservative Clostridial toxin enzymatic domain variant is Clostridial toxin enzymatic domain on which the Clostridial based, that possess at least 50% amino acid identity, 65% toxin enzymatic domain isoform is based, and can be substi amino acid identity, 75% amino acid identity, 85% amino acid tuted for the reference Clostridial toxin enzymatic domain in identity or 95% amino acid identity to the reference any aspect of the present invention. Clostridial toxin enzymatic domain on which the conserva 0024. Another non-limiting examples of a naturally occur tive Clostridial toxin enzymatic domain variant is based. ring Clostridial toxin enzymatic domain variant is a Non-limiting examples of a conservative Clostridial toxin Clostridial toxin enzymatic domain Subtype such as, e.g., an enzymatic domain variant include, e.g., conservative enzymatic domain from subtype BoNT/A1. BoNT/A2, BoNT/A enzymatic domain variants, conservative BoNT/B BoNT/A3, BoNT/A4 and BoNT/A5; an enzymatic domain enzymatic domain variants, conservative BoNT/C1 enzy from subtype BoNT/B1, BoNT/B2, BoNT/B bivalent and matic domain variants, conservative BoNT/D enzymatic BoNT/B nonproteolytic; an enzymatic domain from subtype domain variants, conservative BoNT/E enzymatic domain BoNT/C1-1 and BoNT/C1-2; an enzymatic domain from variants, conservative BoNT/F enzymatic domain variants, subtype BoNT/E1, BoNT/E2 and BoNT/E3; and an enzy conservative BoNT/G enzymatic domain variants, and con matic domain from subtype BoNT/F1, BoNT/F2, BoNT/F3 servative TeNT enzymatic domain variants. and BoNT/F4. A Clostridial toxin enzymatic domain subtype 0027. As used herein, the term “non-conservative can function in Substantially the same manner as the reference Clostridial toxin enzymatic domain variant’ means a Clostridial toxin enzymatic domain on which the Clostridial Clostridial toxin enzymatic domain in which 1) at least one toxin enzymatic domain Subtype is based, and can be substi amino acid is deleted from the reference Clostridial toxin tuted for the reference Clostridial toxin enzymatic domain in enzymatic domain on which the non-conservative Clostridial any aspect of the present invention. toxin enzymatic domain variant is based; 2) at least one amino 0025. As used herein, the term “non-naturally occurring acid added to the reference Clostridial toxin enzymatic Clostridial toxin enzymatic domain variant’ means any domain on which the non-conservative Clostridial toxin Clostridial toxin enzymatic domain produced with the aid of enzymatic domain is based; or 3) at least one amino acid is human manipulation, including, without limitation, Substituted by another amino acid or an amino acid analog Clostridial toxin enzymatic domains produced by genetic that does not share any property similar to that of the original engineering using random mutagenesis or rational design and amino acid from the reference Clostridial toxin enzymatic Clostridial toxin enzymatic domains produced by chemical domain sequence (Table 1). A non-conservative Clostridial synthesis. Non-limiting examples of non-naturally occurring toxin enzymatic domain variant can function in Substantially Clostridial toxin enzymatic domain variants include, e.g., the same manner as the reference Clostridial toxin enzymatic conservative Clostridial toxin enzymatic domain variants, domain on which the non-conservative Clostridial toxin non-conservative Clostridial toxin enzymatic domain vari enzymatic domain variant is based, and can be substituted for ants, Clostridial toxin enzymatic domain chimeric variants the reference Clostridial toxin enzymatic domain in any and active Clostridial toxin enzymatic domain fragments. aspect of the present invention. A non-conservative 0026. As used herein, the term “conservative Clostridial Clostridial toxin enzymatic domain variant can delete one or toxin enzymatic domain variant’ means a Clostridial toxin more amino acids, two or more amino acids, three or more enzymatic domain that has at least one amino acid Substituted amino acids, four or more amino acids, five or more amino by anotheramino acid oranamino acid analog that has at least acids, and ten or more amino acids from the reference one property similar to that of the original amino acid from Clostridial toxin enzymatic domain on which the non-conser the reference Clostridial toxin enzymatic domain sequence Vative Clostridial toxin enzymatic domain variant is based. A (Table 1). Examples of properties include, without limitation, non-conservative Clostridial toxin enzymatic domain variant similar size, topography, charge, hydrophobicity, hydrophi can add one or more amino acids, two or more amino acids, licity, lipophilicity, covalent-bonding capacity, hydrogen three or more amino acids, four or more amino acids, five or bonding capacity, a physicochemical property, of the like, or more amino acids, and ten or more amino acids to the refer any combination thereof. A conservative Clostridial toxin ence Clostridial toxin enzymatic domain on which the non enzymatic domain variant can function in Substantially the conservative Clostridial toxin enzymatic domain variant is same manner as the reference Clostridial toxin enzymatic based. A non-conservative Clostridial toxin enzymatic domain on which the conservative Clostridial toxin enzy domain variant may substitute one or more amino acids, two matic domain variant is based, and can be substituted for the or more amino acids, three or more amino acids, four or more reference Clostridial toxin enzymatic domain in any aspect of amino acids, five or more amino acids, ten or more amino the present invention. A conservative Clostridial toxin enzy acids, 20 or more amino acids, 30 or more amino acids, 40 or matic domain variant may substitute one or more amino more amino acids, 50 or more amino acids, 100 or more acids, two or more amino acids, three or more amino acids, amino acids, or 200 or more amino acids from the reference four or more amino acids, five or more amino acids, ten or Clostridial toxin enzymatic domain on which the non-conser more amino acids, 20 or more amino acids, 30 or more amino Vative Clostridial toxin enzymatic domain variant is based. A US 2009/01171.57 A1 May 7, 2009
non-conservative Clostridial toxin enzymatic domain variant 417-448 of SEQ ID NO: 1) are not required for enzymatic can also substitute at least 10 contiguous amino acids, at least activity. As another non-limiting example, the last 31 amino 15 contiguous amino acids, at least 20 contiguous amino acids of the TeNT enzymatic domain (residues 427-457 of acids, or at least 25 contiguous amino acids from the refer SEQID NO: 8) are not required for enzymatic activity. Thus, ence Clostridial toxin enzymatic domain on which the non aspects of this embodiment can include Clostridial toxin conservative Clostridial toxin enzymatic domain variant is enzymatic domains comprising an enzymatic domain having based, that possess at least 50% amino acid identity, 65% a length of e.g., at least 350 amino acids, at least 375 amino amino acid identity, 75% amino acid identity, 85% amino acid acids, at least 400 amino acids, at least 425 amino acids and at identity or 95% amino acid identity to the reference least 450 amino acids. Other aspects of this embodiment can Clostridial toxin enzymatic domain on which the non-conser include Clostridial toxin enzymatic domains comprising an Vative Clostridial toxin enzymatic domain variant is based. enzymatic domain having a length of e.g., at most 350 amino Non-limiting examples of a non-conservative Clostridial acids, at most 375 amino acids, at most 400 amino acids, at toxin enzymatic domain variant include, e.g., non-conserva most 425 amino acids and at most 450 amino acids. tive BoNT/A enzymatic domain variants, non-conservative 0030. Any of a variety of sequence alignment methods can BoNT/B enzymatic domain variants, non-conservative be used to determine percent identity of naturally-occurring BoNT/C1 enzymatic domain variants, non-conservative Clostridial toxin enzymatic domain variants and non-natu BoNT/D enzymatic domain variants, non-conservative rally-occurring Clostridial toxin enzymatic domain variants, BoNT/E enzymatic domain variants, non-conservative including, without limitation, global methods, local methods BoNT/F enzymatic domain variants, non-conservative and hybrid methods, such as, e.g., segment approach meth BoNT/G enzymatic domain variants, and non-conservative ods. Protocols to determine percent identity are routine pro TeNT enzymatic domain variants. cedures within the scope of one skilled in the art and from the 0028. As used herein, the term “Clostridial toxin enzy teaching herein. matic domain chimeric' means a polypeptide comprising at 0031 Global methods align sequences from the beginning least a portion of a Clostridial toxin enzymatic domain and at to the end of the molecule and determine the best alignment least a portion of at least one other polypeptide to formatoxin by adding up scores of individual residue pairs and by impos enzymatic domain with at least one property different from ing gap penalties. Non-limiting methods include, e.g., the reference Clostridial toxin enzymatic domains of Table 1, CLUSTALW. See, e.g., Julie D. Thompson et al., CLUSTAL with the proviso that this Clostridial toxin enzymatic domain W: Improving the Sensitivity of Progressive Multiple chimeric is still capable of specifically targeting the core Sequence Alignment Through Sequence Weighting, Position components of the neurotransmitter release apparatus and Specific Gap Penalties and Weight Matrix Choice, 22(22) thus participate in executing the overall cellular mechanism Nucleic Acids Research 4673-4680 (1994); and iterative whereby a Clostridial toxin proteolytically cleaves a sub refinement, see, e.g., Osamu Gotoh, Significant Improvement strate. Such Clostridial toxin enzymatic domain chimerics are in Accuracy of Multiple Protein Sequence Alignments by described in, e.g., Lance E. Steward et al., Leucine-based Iterative Refinement as Assessed by Reference to Structural Motif and Clostridial Toxins, U.S. Patent Publication 2003/ Alignments, 264(4) J. Mol. Biol. 823-838 (1996). 0027752 (Feb. 6, 2003); Lance E. Steward et al., Clostridial 0032 Local methods align sequences by identifying one Neurotoxin Compositions and Modified Clostridial Neuro or more conserved motifs shared by all of the input toxins, U.S. Patent Publication 2003/0219462 (Nov. 27, sequences. Non-limiting methods include, e.g., Match-box, 2003); and Lance E. Steward et al., Clostridial Neurotoxin see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box'. A Compositions and Modified Clostridial Neurotoxins, U.S. Fundamentally New Algorithm for the Simultaneous Align Patent Publication 2004/0220386 (Nov. 4, 2004), each of ment of Several Protein Sequences, 8(5) CABIOS 501-509 which is incorporated by reference in its entirety. (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., 0029. As used herein, the term “active Clostridial toxin Detecting Subtle Sequence Signals: A Gibbs Sampling Strat enzymatic domain fragment’ means any of a variety of egy for Multiple Alignment, 262(5131) Science 208-214 Clostridial toxin fragments comprising the enzymatic (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M A domain can be useful in aspects of the present invention with New Algorithm for Multiple Alignment of Highly Divergent the proviso that these enzymatic domain fragments can spe Sequences, 2009) Bioinformatics, 1428-1435 (2004). cifically target the core components of the neurotransmitter 0033 Hybrid methods combine functional aspects of both release apparatus and thus participate in executing the overall global and local alignment methods. Non-limiting methods cellular mechanism whereby a Clostridial toxin proteolyti include, e.g., segment-to-segment comparison, See, e.g., cally cleaves a substrate. The enzymatic domains of Burkhard Morgenstern et al., Multiple DNA and Protein Clostridial toxins are approximately 420-460 amino acids in Sequence Alignment Based On Segment-to-Segment Com length and comprise an enzymatic domain (Table 1). parison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 Research has shown that the entire length of a Clostridial (1996); T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee. toxin enzymatic domain is not necessary for the enzymatic A Novel Algorithm for Multiple Sequence Alignment, 302(1) activity of the enzymatic domain. As a non-limiting example, J. Mol. Biol. 205-217 (2000); MUSCLE, see, e.g., Robert C. the first eight amino acids of the BoNT/A enzymatic domain Edgar, MUSCLE: Multiple Sequence Alignment With High (residues 1-8 of SEQ ID NO: 1) are not required for enzy Score Accuracy and High Throughput, 32(5) Nucleic Acids matic activity. As another non-limiting example, the first Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amaren eight amino acids of the TeNT enzymatic domain (residues dran R Subramanian et al., DIALIGN-T: An Improved Algo 1-8 of SEQID NO: 8) are not required for enzymatic activity. rithm for Segment-Based Multiple Sequence Alignment, 6(1) Likewise, the carboxyl-terminus of the enzymatic domain is BMC Bioinformatics 66 (2005). not necessary for activity. As a non-limiting example, the last 0034. Thus, in an embodiment, a modified Clostridial 32 amino acids of the BoNT/A enzymatic domain (residues toxin disclosed in the present specification comprises a US 2009/01171.57 A1 May 7, 2009
Clostridial toxin enzymatic domain. In an aspect of this acid additions relative to amino acids 1-448 of SEQID NO: 1; embodiment, a Clostridial toxin enzymatic domain com or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 prises a naturally occurring Clostridial toxin enzymatic non-contiguous amino acid additions relative to amino acids domain variant, Such as, e.g., a Clostridial toxin enzymatic 1-448 of SEQID NO: 1. domain isoform or a Clostridial toxin enzymatic domain Sub 0038. In other aspects of this embodiment, a BoNT/A type. In another aspect of this embodiment, a Clostridial toxin enzymatic domain comprises a polypeptide having, e.g., at enzymatic domain comprises a non-naturally occurring most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 Clostridial toxin enzymatic domain variant, such as, e.g., a contiguous amino acid Substitutions relative to amino acids conservative Clostridial toxin enzymatic domain variant, a 1-448 of SEQID NO: 1; at least 1,2,3,4,5,6,7,8,9, 10, 20, non-conservative Clostridial toxin enzymatic domain variant, 30, 40, 50, or 100 contiguous amino acid substitutions relative a Clostridial toxin chimeric enzymatic domain, an active to amino acids 1-448 of SEQID NO: 1; at most 1, 2, 3, 4, 5, Clostridial toxin enzymatic domain fragment, or any combi 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid nation thereof. deletions relative to amino acids 1-448 of SEQID NO: 1; at 0035. In another embodiment, a Clostridial toxin enzy least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 matic domain comprises a BoNT/A enzymatic domain. In an contiguous amino aciddeletions relative to amino acids 1-448 aspect of this embodiment, a BoNT/A enzymatic domain of SEQID NO: 1; at most 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, comprises amino acids 1-448 of SEQ ID NO: 1. In another 50, or 100 contiguous amino acid additions relative to amino aspect of this embodiment, a BoNT/A enzymatic domain acids 1-448 of SEQID NO: 1; or at least 1, 2, 3, 4, 5, 6, 7, 8, comprises a naturally occurring BoNT/A enzymatic domain 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions variant, Such as, e.g., an enzymatic domain from a BoNT/A relative to amino acids 1-448 of SEQID NO: 1. isoform or an enzymatic domain from a BoNT/A subtype. In 0039. In another embodiment, a Clostridial toxin enzy another aspect of this embodiment, a BoNT/A enzymatic matic domain comprises a BoNT/B enzymatic domain. In an domain comprises amino acids 1-448 of a naturally occurring aspect of this embodiment, a BoNT/B enzymatic domain BoNT/A enzymatic domain variant of SEQID NO: 1, such comprises amino acids 1-441 of SEQ ID NO: 2. In another as, e.g., amino acids 1-448 of a BoNT/A isoform of SEQID aspect of this embodiment, a BoNT/B enzymatic domain NO: 1 or amino acids 1-448 of a BoNT/A subtype of SEQID comprises a naturally occurring BoNT/B enzymatic domain NO: 1. In still another aspect of this embodiment, a BoNT/A variant, Such as, e.g., an enzymatic domain from a BoNT/B enzymatic domain comprises a non-naturally occurring isoform or an enzymatic domain from a BoNT/B subtype. In BoNT/A enzymatic domain variant, such as, e.g., a conser another aspect of this embodiment, a BoNT/B enzymatic Vative BoNT/A enzymatic domain variant, a non-conserva domain comprises amino acids 1-441 of a naturally occurring tive BoNT/A enzymatic domain variant, a BoNT/Achimeric BoNT/B enzymatic domain variant of SEQIDNO: 2, such as, enzymatic domain, an active BoNT/A enzymatic domain e.g., amino acids 1-441 of a BoNT/B isoform of SEQID NO: fragment, or any combination thereof. In still another aspect 2 or amino acids 1-441 of a BoNT/B subtype of SEQID NO: of this embodiment, a BoNT/A enzymatic domain comprises 2. In still another aspect of this embodiment, a BoNT/B amino acids 1-448 of a non-naturally occurring BoNT/A enzymatic domain comprises a non-naturally occurring enzymatic domain variant of SEQ ID NO: 1, such as, e.g., BoNTVB enzymatic domain variant, such as, e.g., a conser amino acids 1-448 of a conservative BoNT/A enzymatic vative BoNT/B enzymatic domain variant, a non-conserva domain variant of SEQ ID NO: 1, amino acids 1-448 of a tive BoNT/B enzymatic domain variant, a BoNT/B chimeric non-conservative BoNT/A enzymatic domain variant of SEQ enzymatic domain, an active BoNT/B enzymatic domain ID NO: 1, amino acids 1-448 of an active BoNT/A enzymatic fragment, or any combination thereof. In still another aspect domain fragment of SEQ ID NO: 1, or any combination of this embodiment, a BoNT/B enzymatic domain comprises thereof. amino acids 1-441 of a non-naturally occurring BoNT/B 0036. In other aspects of this embodiment, a BoNT/A enzymatic domain variant of SEQ ID NO: 2, such as, e.g., enzymatic domain comprises a polypeptide having an amino amino acids 1-441 of a conservative BoNT/B enzymatic acid identity to amino acids 1-448 of SEQID NO: 1 of, e.g., domain variant of SEQ ID NO: 2, amino acids 1-441 of a at least 70%, at least 75%, at least 80%, at least 85%, at least non-conservative BoNT/B enzymatic domain variant of SEQ 90% or at least 95%. In yet other aspects of this embodiment, ID NO: 2, amino acids 1-441 of an active BoNT/B enzymatic a BoNT/A enzymatic domain comprises a polypeptide hav domain fragment of SEQ ID NO: 2, or any combination ing an amino acid identity to amino acids 1-448 of SEQ ID thereof. NO: 1 of, e.g., at most 70%, at most 75%, at most 80%, at 0040. In other aspects of this embodiment, a BoNT/B most 85%, at most 90% or at most 95%. enzymatic domain comprises a polypeptide having an amino 0037. In other aspects of this embodiment, a BoNT/A acid identity to amino acids 1-441 of SEQID NO: 2 of, e.g., enzymatic domain comprises a polypeptide having, e.g., at at least 70%, at least 75%, at least 80%, at least 85%, at least most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 90% or at least 95%. In yet other aspects of this embodiment, non-contiguous amino acid substitutions relative to amino a BoNT/B enzymatic domain comprises a polypeptidehaving acids 1-448 of SEQID NO: 1; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, an amino acid identity to amino acids 1-441 of SEQID NO: 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substi 2 of, e.g., at most 70%, at most 75%, at most 80%, at most tutions relative to amino acids 1-448 of SEQ ID NO: 1; at 85%, at most 90% or at most 95%. most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 0041. In other aspects of this embodiment, a BoNT/B non-contiguous amino acid deletions relative to amino acids enzymatic domain comprises a polypeptide having, e.g., at 1-448 of SEQID NO: 1; at least 1,2,3,4,5,6,7,8,9, 10, 20, most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 30, 40, 50, or 100 non-contiguous amino acid deletions rela non-contiguous amino acid substitutions relative to amino tive to amino acids 1-448 of SEQID NO: 1; at most 1, 2, 3, 4, acids 1-441 of SEQID NO: 2; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substi US 2009/01171.57 A1 May 7, 2009
tutions relative to amino acids 1-441 of SEQ ID NO: 2; at NO: 3 of, e.g., at most 70%, at most 75%, at most 80%, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 most 85%, at most 90% or at most 95%. non-contiguous amino acid deletions relative to amino acids 0045. In other aspects of this embodiment, a BoNT/C1 1-441 of SEQID NO: 2; at least 1,2,3,4,5,6,7,8,9, 10, 20, enzymatic domain comprises a polypeptide having, e.g., at 30, 40, 50, or 100 non-contiguous amino acid deletions rela most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 tive to amino acids 1-441 of SEQID NO: 2; at most 1, 2, 3, 4, non-contiguous amino acid substitutions relative to amino 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acids 1-449 of SEQID NO:3: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, acid additions relative to amino acids 1-441 of SEQID NO: 2; 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substi or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 tutions relative to amino acids 1-449 of SEQ ID NO: 3; at non-contiguous amino acid additions relative to amino acids most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 1-441 of SEQID NO: 2. non-contiguous amino acid deletions relative to amino acids 1-449 of SEQID NO:3: at least 1,2,3,4,5,6,7,8,9, 10, 20, 0042. In other aspects of this embodiment, a BoNT/B 30, 40, 50, or 100 non-contiguous amino acid deletions rela enzymatic domain comprises a polypeptide having, e.g., at tive to amino acids 1-449 of SEQID NO:3: at most 1, 2, 3, 4, most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino contiguous amino acid Substitutions relative to amino acids acid additions relative to amino acids 1-449 of SEQID NO:3: 1-441 of SEQID NO: 2; at least 1,2,3,4,5,6,7,8,9, 10, 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 30, 40, 50, or 100 contiguous amino acid substitutions relative non-contiguous amino acid additions relative to amino acids to amino acids 1-441 of SEQID NO: 2; at most 1, 2, 3, 4, 5, 1-449 of SEQID NO: 3. 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid 0046. In other aspects of this embodiment, a BoNT/C1 deletions relative to amino acids 1-441 of SEQID NO: 2; at enzymatic domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions relative to amino acids 1-441 contiguous amino acid Substitutions relative to amino acids of SEQID NO:2; at most 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 1-449 of SEQID NO:3: at least 1,2,3,4,5,6,7,8,9, 10, 20, 50, or 100 contiguous amino acid additions relative to amino 30, 40, 50, or 100 contiguous amino acid substitutions relative acids 1-441 of SEQID NO: 2; or at least 1, 2, 3, 4, 5, 6, 7, 8, to amino acids 1-449 of SEQID NO: 3; at most 1, 2, 3, 4, 5, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid relative to amino acids 1-441 of SEQID NO: 2. deletions relative to amino acids 1-449 of SEQID NO: 3; at 0043. In another embodiment, a Clostridial toxin enzy least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 matic domain comprises a BoNT/C1 enzymatic domain. In an contiguous amino aciddeletions relative to amino acids 1-449 aspect of this embodiment, a BoNT/C1 enzymatic domain of SEQID NO:3: at most 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, comprises amino acids 1-449 of SEQ ID NO: 3. In another 50, or 100 contiguous amino acid additions relative to amino aspect of this embodiment, a BoNT/C1 enzymatic domain acids 1-449 of SEQID NO:3: or at least 1, 2, 3, 4, 5, 6, 7, 8, comprises a naturally occurring BoNT/C1 enzymatic domain 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions variant, Such as, e.g., an enzymatic domain from a BoNT/C1 relative to amino acids 1-449 of SEQID NO:3. isoform or an enzymatic domain from a BoNT/C1 subtype. In 0047. In another embodiment, a Clostridial toxin enzy another aspect of this embodiment, a BoNT/C1 enzymatic matic domain comprises a BoNT/D enzymatic domain. In an domain comprises amino acids 1-449 of a naturally occurring aspect of this embodiment, a BoNTVD enzymatic domain BoNT/C1 enzymatic domain variant of SEQID NO:3, such comprises amino acids 1-445 of SEQ ID NO: 4. In another as, e.g., amino acids 1-449 of a BoNT/C1 isoform of SEQID aspect of this embodiment, a BoNTVD enzymatic domain NO:3 or amino acids 1-449 of a BoNT/C1 subtype of SEQID comprises a naturally occurring BoNTVD enzymatic domain NO:3. In still another aspect of this embodiment, a BoNT/C1 variant, such as, e.g., an enzymatic domain from a BoNTVD enzymatic domain comprises a non-naturally occurring isoform or an enzymatic domain from a BoNT/D subtype. In BoNT/C1 enzymatic domain variant, such as, e.g., a conser another aspect of this embodiment, a BoNT/D enzymatic vative BoNT/C1 enzymatic domain variant, a non-conserva domain comprises amino acids 1-445 of a naturally occurring tive BoNT/C1 enzymatic domain variant, a BoNT/C1 chi BoNT/D enzymatic domain variant of SEQID NO: 4, such meric enzymatic domain, an active BoNT/C1 enzymatic as, e.g., amino acids 1-445 of a BoNT/D isoform of SEQID domain fragment, or any combination thereof. In still another NO. 4 or amino acids 1-445 of a BoNT/D subtype of SEQID aspect of this embodiment, a BoNT/C1 enzymatic domain NO: 4. In still another aspect of this embodiment, a BoNT/D comprises amino acids 1-449 of a non-naturally occurring enzymatic domain comprises a non-naturally occurring BoNT/C1 enzymatic domain variant of SEQID NO:3, such BoNTVD enzymatic domain variant, such as, e.g., a conser as, e.g., amino acids 1-449 of a conservative BoNT/C1 enzy vative BoNT/D enzymatic domain variant, a non-conserva matic domain variant of SEQID NO:3, amino acids 1-449 of tive BoNT/D enzymatic domain variant, a BoNT/D chimeric a non-conservative BoNT/C1 enzymatic domain variant of enzymatic domain, an active BoNTVD enzymatic domain SEQ ID NO: 3, amino acids 1-449 of an active BoNT/C1 fragment, or any combination thereof. In still another aspect enzymatic domain fragment of SEQID NO:3, or any com of this embodiment, a BoNT/D enzymatic domain comprises bination thereof. amino acids 1-445 of a non-naturally occurring BoNT/D 0044. In other aspects of this embodiment, a BoNT/C1 enzymatic domain variant of SEQ ID NO: 4, such as, e.g., enzymatic domain comprises a polypeptide having an amino amino acids 1-445 of a conservative BoNT/D enzymatic acid identity to amino acids 1-449 of SEQID NO: 3 of, e.g., domain variant of SEQ ID NO: 4, amino acids 1-445 of a at least 70%, at least 75%, at least 80%, at least 85%, at least non-conservative BoNT/D enzymatic domain variant of SEQ 90% or at least 95%. In yet other aspects of this embodiment, ID NO: 4, amino acids 1-445 of an active BoNT/D enzymatic a BoNT/C1 enzymatic domain comprises a polypeptide hav domain fragment of SEQ ID NO: 4, or any combination ing an amino acid identity to amino acids 1-449 of SEQ ID thereof. US 2009/01171.57 A1 May 7, 2009
0048. In other aspects of this embodiment, a BoNT/D enzymatic domain variant of SEQ ID NO: 5, such as, e.g., enzymatic domain comprises a polypeptide having an amino amino acids 1-422 of a conservative BoNT/E enzymatic acid identity to amino acids 1-445 of SEQID NO. 4 of, e.g., domain variant of SEQ ID NO: 5, amino acids 1-422 of a at least 70%, at least 75%, at least 80%, at least 85%, at least non-conservative BoNT/E enzymatic domain variant of SEQ 90% or at least 95%. In yet other aspects of this embodiment, ID NO: 5, amino acids 1-422 of an active BoNT/E enzymatic a BoNT/D enzymatic domain comprises a polypeptide hav domain fragment of SEQ ID NO: 5, or any combination ing an amino acid identity to amino acids 1-445 of SEQ ID thereof. NO. 4 of, e.g., at most 70%, at most 75%, at most 80%, at 0052. In other aspects of this embodiment, a BoNT/E most 85%, at most 90% or at most 95%. enzymatic domain comprises a polypeptide having an amino 0049. In other aspects of this embodiment, a BoNT/D acid identity to amino acids 1-422 of SEQID NO: 5 of, e.g., enzymatic domain comprises a polypeptide having, e.g., at at least 70%, at least 75%, at least 80%, at least 85%, at least most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 90% or at least 95%. In yet other aspects of this embodiment, non-contiguous amino acid substitutions relative to amino a BoNT/E enzymatic domain comprises a polypeptide having acids 1-445 of SEQID NO: 4; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, an amino acid identity to amino acids 1-422 of SEQID NO: 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substi 5 of, e.g., at most 70%, at most 75%, at most 80%, at most tutions relative to amino acids 1-445 of SEQ ID NO: 4; at 85%, at most 90% or at most 95%. most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 0053. In other aspects of this embodiment, a BoNT/E non-contiguous amino acid deletions relative to amino acids enzymatic domain comprises a polypeptide having, e.g., at 1-445 of SEQID NO: 4; at least 1,2,3,4,5,6,7,8,9, 10, 20, most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 30, 40, 50, or 100 non-contiguous amino acid deletions rela non-contiguous amino acid substitutions relative to amino tive to amino acids 1-445 of SEQID NO: 4; at most 1, 2, 3, 4, acids 1-422 of SEQID NO: 5; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substi acid additions relative to amino acids 1-445 of SEQID NO: 4; tutions relative to amino acids 1-422 of SEQ ID NO: 5; at or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid additions relative to amino acids non-contiguous amino acid deletions relative to amino acids 1-445 of SEQID NO: 4. 1-422 of SEQID NO: 5; at least 1,2,3,4,5,6,7,8,9, 10, 20, 0050. In other aspects of this embodiment, a BoNT/D 30, 40, 50, or 100 non-contiguous amino acid deletions rela enzymatic domain comprises a polypeptide having, e.g., at tive to amino acids 1-422 of SEQID NO: 5; at most 1, 2, 3, 4, most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino contiguous amino acid Substitutions relative to amino acids acid additions relative to amino acids 1-422 of SEQID NO: 5; 1-445 of SEQID NO: 4; at least 1,2,3,4,5,6,7,8,9, 10, 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 30, 40, 50, or 100 contiguous amino acid substitutions relative non-contiguous amino acid additions relative to amino acids to amino acids 1-445 of SEQID NO: 4; at most 1, 2, 3, 4, 5, 1-422 of SEQID NO: 5. 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid 0054) In other aspects of this embodiment, a BoNT/E deletions relative to amino acids 1-445 of SEQID NO: 4; at enzymatic domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions relative to amino acids 1-445 contiguous amino acid Substitutions relative to amino acids of SEQID NO: 4; at most 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 1-422 of SEQID NO: 5; at least 1,2,3,4,5,6,7,8,9, 10, 20, 50, or 100 contiguous amino acid additions relative to amino 30, 40, 50, or 100 contiguous amino acid substitutions relative acids 1-445 of SEQID NO: 4; or at least 1, 2, 3, 4, 5, 6, 7, 8, to amino acids 1-422 of SEQID NO: 5; at most 1, 2, 3, 4, 5, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid relative to amino acids 1-445 of SEQID NO: 4. deletions relative to amino acids 1-422 of SEQID NO: 5; at 0051. In another embodiment, a Clostridial toxin enzy least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 matic domain comprises a BoNT/E enzymatic domain. In an contiguous amino aciddeletions relative to amino acids 1-422 aspect of this embodiment, a BoNT/E enzymatic domain of SEQID NO: 5; at most 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, comprises amino acids 1-422 of SEQ ID NO: 5. In another 50, or 100 contiguous amino acid additions relative to amino aspect of this embodiment, a BoNT/E enzymatic domain acids 1-422 of SEQID NO: 5; or at least 1, 2, 3, 4, 5, 6, 7, 8, comprises a naturally occurring BoNT/E enzymatic domain 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions variant, such as, e.g., an enzymatic domain from a BoNT/E relative to amino acids 1-422 of SEQID NO: 5. isoform or an enzymatic domain from a BoNT/E subtype. In 0055. In another embodiment, a Clostridial toxin enzy another aspect of this embodiment, a BoNT/E enzymatic matic domain comprises a BoNT/F enzymatic domain. In an domain comprises amino acids 1-422 of a naturally occurring aspect of this embodiment, a BoNT/F enzymatic domain BoNT/E enzymatic domain variant of SEQID NO: 5, such as, comprises amino acids 1-439 of SEQ ID NO: 6. In another e.g., amino acids 1-422 of a BoNT/E isoform of SEQID NO: aspect of this embodiment, a BoNT/F enzymatic domain 5 or amino acids 1-422 of a BoNT/E subtype of SEQID NO: comprises a naturally occurring BoNT/F enzymatic domain 5. In still another aspect of this embodiment, a BoNT/E enzy variant, such as, e.g., an enzymatic domain from a BoNT/F matic domain comprises a non-naturally occurring BoNT/E isoform or an enzymatic domain from a BoNT/F Subtype. In enzymatic domain variant, such as, e.g., a conservative another aspect of this embodiment, a BoNT/F enzymatic BoNT/E enzymatic domain variant, a non-conservative domain comprises amino acids 1-439 of a naturally occurring BoNT/E enzymatic domain variant, a BoNT/E chimeric BoNT/F enzymatic domain variant of SEQID NO: 6, such as, enzymatic domain, an active BoNT/E enzymatic domain e.g., amino acids 1-439 of a BoNT/F isoform of SEQID NO: fragment, or any combination thereof. In still another aspect 6 or amino acids 1-439 of a BoNT/F subtype of SEQID NO: of this embodiment, a BoNT/E enzymatic domain comprises 6. In still another aspect of this embodiment, a BoNT/F enzy amino acids 1-422 of a non-naturally occurring BoNT/E matic domain comprises a non-naturally occurring BoNT/F US 2009/01171.57 A1 May 7, 2009
enzymatic domain variant, such as, e.g., a conservative another aspect of this embodiment, a BoNT/G enzymatic BoNT/F enzymatic domain variant, a non-conservative domain comprises amino acids 1-446 of a naturally occurring BoNT/F enzymatic domain variant, a BoNT/F chimeric enzy BoNT/G enzymatic domain variant of SEQ ID NO: 7, such matic domain, an active BoNT/F enzymatic domain frag as, e.g., amino acids 1-446 of a BoNT/G isoform of SEQID ment, or any combination thereof. In still another aspect of NO: 7 or amino acids 1-446 of a BoNT/G subtype of SEQID this embodiment, a BoNT/F enzymatic domain comprises NO: 7. In still another aspect of this embodiment, a BoNT/G amino acids 1-439 of a non-naturally occurring BoNT/F enzymatic domain comprises a non-naturally occurring enzymatic domain variant of SEQ ID NO: 6, such as, e.g., BoNT/G enzymatic domain variant, such as, e.g., a conser amino acids 1-439 of a conservative BoNT/F enzymatic vative BoNT/G enzymatic domain variant, a non-conserva domain variant of SEQ ID NO: 6, amino acids 1-439 of a non-conservative BoNT/F enzymatic domain variant of SEQ tive BoNT/G enzymatic domain variant, a BoNT/G chimeric ID NO: 6, amino acids 1-439 of an active BoNT/F enzymatic enzymatic domain, an active BoNT/G enzymatic domain domain fragment of SEQ ID NO: 6, or any combination fragment, or any combination thereof. In still another aspect thereof. of this embodiment, a BoNT/G enzymatic domain comprises 0056. In other aspects of this embodiment, a BoNT/F amino acids 1-446 of a non-naturally occurring BoNT/G enzymatic domain comprises a polypeptide having an amino enzymatic domain variant of SEQ ID NO: 7, such as, e.g., acid identity to amino acids 1-439 of SEQID NO: 6 of, e.g., amino acids 1-446 of a conservative BoNT/G enzymatic at least 70%, at least 75%, at least 80%, at least 85%, at least domain variant of SEQ ID NO: 7, amino acids 1-446 of a 90% or at least 95%. In yet other aspects of this embodiment, non-conservative BoNT/G enzymatic domain variant of SEQ a BoNT/F enzymatic domain comprises a polypeptide having ID NO: 7, amino acids 1-446 of an active BoNT/G enzymatic an amino acid identity to amino acids 1-439 of SEQID NO: domain fragment of SEQ ID NO: 7, or any combination 6 of, e.g., at most 70%, at most 75%, at most 80%, at most thereof. 85%, at most 90% or at most 95%. 0060. In other aspects of this embodiment, a BoNT/G 0057. In other aspects of this embodiment, a BoNT/F enzymatic domain comprises a polypeptide having an amino enzymatic domain comprises a polypeptide having, e.g., at acid identity to amino acids 1-446 of SEQID NO: 7 of, e.g., most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 at least 70%, at least 75%, at least 80%, at least 85%, at least non-contiguous amino acid substitutions relative to amino 90% or at least 95%. In yet other aspects of this embodiment, acids 1-439 of SEQID NO: 6; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, a BoNT/G enzymatic domain comprises a polypeptide hav 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substi ing an amino acid identity to amino acids 1-446 of SEQID tutions relative to amino acids 1-439 of SEQ ID NO: 6; at NO: 7 of, e.g., at most 70%, at most 75%, at most 80%, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 most 85%, at most 90% or at most 95%. non-contiguous amino acid deletions relative to amino acids 0061. In other aspects of this embodiment, a BoNT/G 1-439 of SEQID NO: 6; at least 1,2,3,4,5,6,7,8,9, 10, 20, enzymatic domain comprises a polypeptide having, e.g., at 30, 40, 50, or 100 non-contiguous amino acid deletions rela most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 tive to amino acids 1-439 of SEQID NO: 6; at most 1, 2, 3, 4, non-contiguous amino acid substitutions relative to amino 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acids 1-446 of SEQID NO: 7; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, acid additions relative to amino acids 1-439 of SEQID NO: 6; 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substi or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 tutions relative to amino acids 1-446 of SEQ ID NO: 7; at non-contiguous amino acid additions relative to amino acids most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 1-439 of SEQID NO: 6. non-contiguous amino acid deletions relative to amino acids 0058. In other aspects of this embodiment, a BoNT/F 1-446 of SEQID NO: 7; at least 1,2,3,4,5,6,7,8,9, 10, 20, enzymatic domain comprises a polypeptide having, e.g., at 30, 40, 50, or 100 non-contiguous amino acid deletions rela most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 tive to amino acids 1-446 of SEQID NO: 7; at most 1, 2, 3, 4, contiguous amino acid Substitutions relative to amino acids 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino 1-439 of SEQID NO: 6; at least 1,2,3,4,5,6,7,8,9, 10, 20, acid additions relative to amino acids 1-446 of SEQID NO:7; 30, 40, 50, or 100 contiguous amino acid substitutions relative or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 to amino acids 1-439 of SEQID NO: 6; at most 1, 2, 3, 4, 5, non-contiguous amino acid additions relative to amino acids 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid 1-446 of SEQID NO: 7. deletions relative to amino acids 1-439 of SEQID NO: 6; at 0062. In other aspects of this embodiment, a BoNT/G least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 enzymatic domain comprises a polypeptide having, e.g., at contiguous amino aciddeletions relative to amino acids 1-439 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 of SEQID NO: 6; at most 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, contiguous amino acid Substitutions relative to amino acids 50, or 100 contiguous amino acid additions relative to amino 1-446 of SEQID NO: 7; at least 1,2,3,4,5,6,7,8,9, 10, 20, acids 1-439 of SEQID NO: 6; or at least 1, 2, 3, 4, 5, 6, 7, 8, 30, 40, 50, or 100 contiguous amino acid substitutions relative 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions to amino acids 1-446 of SEQID NO: 7; at most 1, 2, 3, 4, 5, relative to amino acids 1-439 of SEQID NO: 6. 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid 0059. In another embodiment, a Clostridial toxin enzy deletions relative to amino acids 1-446 of SEQID NO: 7; at matic domain comprises a BoNT/G enzymatic domain. In an least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 aspect of this embodiment, a BoNT/G enzymatic domain contiguous amino aciddeletions relative to amino acids 1-446 comprises amino acids 1-446 of SEQ ID NO: 7. In another of SEQID NO: 7; at most 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, aspect of this embodiment, a BoNT/G enzymatic domain 50, or 100 contiguous amino acid additions relative to amino comprises a naturally occurring BoNT/G enzymatic domain acids 1-446 of SEQID NO: 7; or at least 1, 2, 3, 4, 5, 6, 7, 8, variant, Such as, e.g., an enzymatic domain from a BoNT/G 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions isoform or an enzymatic domain from a BoNT/G subtype. In relative to amino acids 1-446 of SEQID NO: 7. US 2009/01171.57 A1 May 7, 2009
0063. In another embodiment, a Clostridial toxin enzy amino acid additions relative to amino acids 1-457 of SEQID matic domain comprises a TeNT enzymatic domain. In an NO: 8; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or aspect of this embodiment, a TeNT enzymatic domain com 100 contiguous amino acid additions relative to amino acids prises amino acids 1-457 of SEQID NO: 8. In another aspect 1-457 of SEQID NO:8. of this embodiment, a TeNT enzymatic domain comprises a 0067. In another embodiment, a Clostridial toxin enzy naturally occurring TeNT enzymatic domain variant, such as, matic domain comprises a BaNT enzymatic domain. In an e.g., an enzymatic domain from a TeNT isoform or an enzy aspect of this embodiment, a BaNT enzymatic domain com matic domain from a TeNT subtype. In another aspect of this prises amino acids 1-431 of SEQID NO:9. In another aspect embodiment, a TeNT enzymatic domain comprises amino of this embodiment, a BaNT enzymatic domain comprises a acids 1-457 of a naturally occurring TeNT enzymatic domain naturally occurring BaNT enzymatic domain variant, Such as, variant of SEQID NO: 8, such as, e.g., amino acids 1-457 of e.g., an enzymatic domain from a BaNT isoform or an enzy a TeNT isoform of SEQID NO: 8 or amino acids 1-457 of a matic domain from a BaNT subtype. In another aspect of this TeNT subtype of SEQID NO: 8. In still another aspect of this embodiment, a BaNT enzymatic domain comprises amino embodiment, a TeNT enzymatic domain comprises a non acids 1-431 of a naturally occurring BaNT enzymatic domain naturally occurring TeNT enzymatic domain variant, such as, variant of SEQID NO:9, such as, e.g., amino acids 1-431 of e.g., a conservative TeNT enzymatic domain variant, a non a BaNT isoform of SEQID NO:9 or amino acids 1-431 of a conservative TeNT enzymatic domain variant, a TeNT chi BaNT subtype of SEQID NO:9. In still another aspect of this meric enzymatic domain, an active TeNT enzymatic domain embodiment, a BaNT enzymatic domain comprises a non fragment, or any combination thereof. In still another aspect naturally occurring BaNT enzymatic domain variant, Such as, of this embodiment, a TeNT enzymatic domain comprises e.g., a conservative BaNT enzymatic domain variant, a non amino acids 1-457 of a non-naturally occurring TeNT enzy conservative BaNT enzymatic domain variant, a BaNT chi matic domain variant of SEQID NO: 8, such as, e.g., amino meric enzymatic domain, an active BaNT enzymatic domain acids 1-457 of a conservative TeNT enzymatic domain vari fragment, or any combination thereof. In still another aspect ant of SEQID NO: 8, amino acids 1-457 of a non-conserva of this embodiment, a BaNT enzymatic domain comprises tive TeNT enzymatic domain variant of SEQID NO: 8, amino amino acids 1-431 of a non-naturally occurring BaNT enzy acids 1-457 of an active TeNT enzymatic domain fragment of matic domain variant of SEQID NO: 9, such as, e.g., amino SEQID NO: 8, or any combination thereof. acids 1-431 of a conservative BaNT enzymatic domain vari 0064. In other aspects of this embodiment, a TeNT enzy ant of SEQID NO: 9, amino acids 1-431 of a non-conserva matic domain comprises a polypeptide having an amino acid tive BaNT enzymatic domain variant of SEQ ID NO: 9, identity to amino acids 1-457 of SEQID NO: 8 of, e.g., at least amino acids 1-431 of an active BaNT enzymatic domain 70%, at least 75%, at least 80%, at least 85%, at least 90% or fragment of SEQID NO:9, or any combination thereof. at least 95%. In yet other aspects of this embodiment, a TeNT 0068. In other aspects of this embodiment, a BaNT enzy enzymatic domain comprises a polypeptide having an amino matic domain comprises a polypeptide having an amino acid acid identity to amino acids 1-457 of SEQID NO: 8 of, e.g., identity to amino acids 1-431 of SEQIDNO:9 of, e.g., at least at most 70%, at most 75%, at most 80%, at most 85%, at most 70%, at least 75%, at least 80%, at least 85%, at least 90% or 90% or at most 95%. at least 95%. In yet other aspects of this embodiment, a BaNT 0065. In other aspects of this embodiment, a TeNT enzy enzymatic domain comprises a polypeptide having an amino matic domain comprises a polypeptide having, e.g., at most 1, acid identity to amino acids 1-431 of SEQID NO: 9 of, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous at most 70%, at most 75%, at most 80%, at most 85%, at most amino acid substitutions relative to amino acids 1-457 of SEQ 90% or at most 95%. ID NO: 8; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 0069. In other aspects of this embodiment, a BaNT enzy 100 non-contiguous amino acid Substitutions relative to matic domain comprises a polypeptidehaving, e.g., at most 1, amino acids 1-457 of SEQID NO: 8; at most 1, 2, 3, 4, 5, 6, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid amino acid substitutions relative to amino acids 1-431 of SEQ deletions relative to amino acids 1-457 of SEQID NO: 8; at ID NO: 9; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 100 non-contiguous amino acid Substitutions relative to non-contiguous amino acid deletions relative to amino acids amino acids 1-431 of SEQID NO: 9; at most 1, 2, 3, 4, 5, 6, 1-457 of SEQID NO: 8; at most 1,2,3,4,5,6,7,8,9, 10, 20, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid 30, 40, 50, or 100 non-contiguous amino acid additions rela deletions relative to amino acids 1-431 of SEQID NO: 9; at tive to amino acids 1-457 of SEQID NO: 8:or at least 1, 2, 3, least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous amino non-contiguous amino acid deletions relative to amino acids acid additions relative to amino acids 1-457 of SEQID NO: 8. 1-431 of SEQID NO: 9; at most 1,2,3,4,5,6,7,8,9, 10, 20, 0066. In other aspects of this embodiment, a TeNT enzy 30, 40, 50, or 100 non-contiguous amino acid additions rela matic domain comprises a polypeptide having, e.g., at most 1, tive to amino acids 1-431 of SEQID NO: 9; or at least 1, 2, 3, 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino 4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid substitutions relative to amino acids 1-457 of SEQ ID acid additions relative to amino acids 1-431 of SEQID NO:9. NO: 8; at least 1, 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 0070. In other aspects of this embodiment, a BaNT enzy contiguous amino acid Substitutions relative to amino acids matic domain comprises a polypeptidehaving, e.g., at most 1, 1-457 of SEQID NO: 8; at most 1,2,3,4,5,6,7,8,9, 10, 20, 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino 30, 40, 50, or 100 contiguous amino acid deletions relative to acid substitutions relative to amino acids 1-431 of SEQ ID amino acids 1-457 of SEQID NO: 8; at least 1,2,3,4,5,6,7, NO: 9; at least 1, 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid dele contiguous amino acid Substitutions relative to amino acids tions relative to amino acids 1-457 of SEQID NO: 8; at most 1-431 of SEQID NO: 9; at most 1,2,3,4,5,6,7,8,9, 10, 20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous 30, 40, 50, or 100 contiguous amino acid deletions relative to US 2009/01171.57 A1 May 7, 2009
amino acids 1-431 of SEQID NO:9; at least 1,2,3,4,5,6,7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or 200 contiguous 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid dele amino acid substitutions relative to amino acids 1-422 of SEQ tions relative to amino acids 1-431 of SEQID NO: 9; at most ID NO: 10. In other aspects of this embodiment, a BuNT 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous enzymatic domain comprises a polypeptide having, e.g., at amino acid additions relative to amino acids 1-431 of SEQID least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or 200 NO: 9; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or contiguous amino acid Substitutions relative to amino acids 100 contiguous amino acid additions relative to amino acids 1-422 of SEQID NO: 10. In yet other aspects of this embodi 1-431 of SEQID NO: 9. ment, a BuNT enzymatic domain comprises a polypeptide 0071. In another embodiment, a Clostridial toxin enzy having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50. matic domain comprises a BuNT enzymatic domain. In an 100 or 200 contiguous amino acid deletions relative to amino aspect of this embodiment, a BuNT enzymatic domain com acids 1-422 of SEQ ID NO: 10. In other aspects of this prises amino acids 1-422 of SEQID NO: 10. In another aspect embodiment, a BuNT enzymatic domain comprises a of this embodiment, a BuNT enzymatic domain comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, naturally occurring BuNT enzymatic domain variant, such as, 30, 40, 50, 100 or 200 contiguous amino acid deletions rela e.g., an enzymatic domain from a BuNT isoform or an enzy tive to amino acids 1-422 of SEQ ID NO: 10. In still other matic domain from a BuNT subtype. In another aspect of this aspects of this embodiment, a BuNT enzymatic domain com embodiment, a BuNT enzymatic domain comprises amino prises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, acids 1-422 of a naturally occurring BuNT enzymatic domain 9, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid variant of SEQID NO: 10, such as, e.g., amino acids 1-422 of additions relative to amino acids 1-422 of SEQID NO: 10. In a BuNT isoform of SEQID NO: 10 or amino acids 1-422 of other aspects of this embodiment, a BuNT enzymatic domain a BuNT subtype of SEQID NO: 10. In still another aspect of comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, this embodiment, a BuNT enzymatic domain comprises a 8, 9, 10, 20, 30, 40, 50, 100 or 200 contiguous amino acid non-naturally occurring BuNT enzymatic domain variant, additions relative to amino acids 1-422 of SEQID NO: 10. Such as, e.g., a conservative BuNT enzymatic domain variant, 0075. The “translocation domain comprises a portion of a a non-conservative BuNT enzymatic domain variant, a BuNT Clostridial neurotoxin heavy chain having a translocation chimeric enzymatic domain, an active BuNT enzymatic activity. By “translocation is meant the ability to facilitate domain fragment, or any combination thereof. In still another the transport of a polypeptide through a vesicular membrane, aspect of this embodiment, a BuNT enzymatic domain com thereby exposing some or all of the polypeptide to the cyto prises amino acids 1-422 of a non-naturally occurring BuNT plasm. In the various botulinum neurotoxins translocation is enzymatic domain variant of SEQID NO: 10, such as, e.g., thought to involve an allosteric conformational change of the amino acids 1-422 of a conservative BuNT enzymatic domain heavy chain caused by a decrease in pH within the endosome. variant of SEQ ID NO: 10, amino acids 1-422 of a non This conformational change appears to involve and be medi conservative BuNT enzymatic domain variant of SEQ ID ated by the N terminal half of the heavy chain and to result in NO: 10, amino acids 1-422 of an active BuNT enzymatic the formation of pores in the vesicular membrane; this change domain fragment of SEQ ID NO: 10, or any combination permits the movement of the proteolytic light chain from thereof. within the endosomal vesicle into the cytoplasm. See e.g., 0072. In other aspects of this embodiment, a BuNT enzy Lacy, et al., Nature Struct. Biol. 5:898-902 (October 1998). matic domain comprises a polypeptide having an amino acid 0076. The amino acid sequence of the translocation-me identity to amino acids 1-422 of SEQID NO: 10 of, e.g., at diating portion of the botulinum neurotoxin heavy chain is least 70%, at least 75%, at least 80%, at least 85%, at least known to those of skill in the art; additionally, those amino 90% or at least 95%. In yet other aspects of this embodiment, acid residues within this portion that are known to be essential a BuNT enzymatic domain comprises a polypeptide having for conferring the translocation activity are also known. It an amino acid identity to amino acids 1-422 of SEQID NO: would therefore be well within the ability of one of ordinary 10 of, e.g., at most 70%, at most 75%, at most 80%, at most skill in the art, for example, to employ the naturally occurring 85%, at most 90% or at most 95%. N-terminal peptide half of the heavy chain of any of the 0073. In other aspects of this embodiment, a BuNT enzy various Clostridium tetanus or Clostridium botulinum neuro matic domain comprises a polypeptide having, e.g., at most 1, toxin Subtypes as a translocation domain, or to design an 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous analogous translocation domain by aligning the primary amino acid substitutions relative to amino acids 1-422 of SEQ sequences of the N-terminal halves of the various heavy ID NO: 1; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or chains and selecting a consensus primary translocation 100 non-contiguous amino acid Substitutions relative to sequence based on conserved amino acid, polarity, Steric and amino acids 1-422 of SEQID NO: 10; at most 1, 2, 3, 4, 5, 6, hydrophobicity characteristics between the sequences. 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid 0077. In another aspect of the invention, a modified deletions relative to amino acids 1-422 of SEQID NO: 10; at Clostridial toxin comprises, in part, a Clostridial toxin trans least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 location domain. As used herein, the term "Clostridial toxin non-contiguous amino acid deletions relative to amino acids translocation domain” means any Clostridial toxin polypep 1-422 of SEQID NO: 10; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, tide that can execute the translocation step of the intoxication 20, 30, 40, 50, or 100 non-contiguous amino acid additions process that mediates Clostridial toxin light chain transloca relative to amino acids 1-422 of SEQID NO: 10; or at least 1, tion. Thus, a Clostridial toxin translocation domain facilitates 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous the movement of a Clostridial toxin light chain across a mem amino acid additions relative to amino acids 1-422 of SEQID brane and encompasses the movement of a Clostridial toxin NO: 1.O. light chain through the membranean intracellular vesicle into 0074. In other aspects of this embodiment, a BuNT enzy the cytoplasm of a cell. Non-limiting examples of a matic domain comprises a polypeptide having, e.g., at most 1, Clostridial toxin translocation domain include, e.g., a US 2009/01171.57 A1 May 7, 2009
BoNT/A translocation domain, a BoNT/B translocation acids 447-865 of SEQID NO: 7 will have at least one amino domain, a BoNT/C1 translocation domain, a BoNTVD trans acid difference. Such as, e.g., an amino acid substitution, location domain, a BoNT/E translocation domain, a BoNT/F deletion or addition, as compared to the amino acid region translocation domain, a BoNT/G translocation domain, a 447-865 of SEQ ID NO: 7; a TeNT translocation domain TeNT translocation domain, a BaNT translocation domain, variant comprising amino acids 458-881 of SEQ ID NO: 8 and a BuNT translocation domain. Other non-limiting will have at least one amino acid difference, Such as, e.g., an examples of a Clostridial toxin translocation domain include, amino acid Substitution, deletion or addition, as compared to e.g., amino acids 449-873 of SEQ ID NO: 1, amino acids the amino acid region 458-881 of SEQ ID NO: 8; a BaNT 442-860 of SEQID NO: 2, amino acids 450-868 of SEQID translocation domain variant comprising amino acids 432 NO:3, amino acids 446-864 of SEQID NO: 4, amino acids 857 of SEQ ID NO:9 will have at least one amino acid 423-847 of SEQID NO: 5, amino acids 440-866 of SEQID difference. Such as, e.g., an amino acid Substitution, deletion NO: 6, amino acids 447-865 of SEQID NO: 7, amino acids or addition, as compared to the amino acid region 432-857 of 458-881 of SEQID NO: 8, amino acids 432-857 of SEQID SEQ ID NO: 9; and a BuNT translocation domain variant NO:9, and amino acids 423-847 of SEQID NO: 10. comprising amino acids 423-847 of SEQID NO: 10 will have 0078 A Clostridial toxin translocation domain includes, at least one amino acid difference, such as, e.g., an amino acid without limitation, naturally occurring Clostridial toxin Substitution, deletion or addition, as compared to the amino translocation domain variants, such as, e.g., Clostridial toxin acid region 423-847 of SEQID NO: 10. translocation domain isoforms and Clostridial toxin translo 0080. It is recognized by those of skill in the art that within cation domain Subtypes; non-naturally occurring Clostridial each serotype of Clostridial toxin there can be naturally toxin translocation domain variants, such as, e.g., conserva occurring Clostridial toxin translocation domain variants that tive Clostridial toxin translocation domain variants, non-con differ somewhat in their amino acid sequence, and also in the servative Clostridial toxin translocation domain variants, nucleic acids encoding these proteins. For example, there are Clostridial toxin translocation domain chimerics, active presently five BoNT/A subtypes, BoNT/A1, BoNT/A2, Clostridial toxin translocation domain fragments thereof, or BoNT/A3, BoNT/A4, and BoNT/A5, with specific translo any combination thereof. cation domain Subtypes showing approximately 87% amino 0079. As used herein, the term “Clostridial toxin translo acid identity when compared to another BoNT/A transloca cation domain variant, whether naturally-occurring or non tion domain subtype. As used herein, the term “naturally naturally-occurring, means a Clostridial toxin translocation occurring Clostridial toxin translocation domain variant' domain that has at least one amino acid change from the means any Clostridial toxin translocation domain produced corresponding region of the disclosed reference sequences by a naturally-occurring process, including, without limita (Table 1) and can be described in percent identity to the tion, Clostridial toxin translocation domain isoforms pro corresponding region of that reference sequence. Unless duced from alternatively-spliced transcripts, Clostridial toxin expressly indicated, Clostridial toxin translocation domain translocation domain isoforms produced by spontaneous variants useful to practice disclosed embodiments are vari mutation and Clostridial toxin translocation domain Sub ants that execute the translocation step of the intoxication types. A naturally occurring Clostridial toxin translocation process that mediates Clostridial toxin light chain transloca domain variant can function in Substantially the same manner tion. As non-limiting examples, a BoNT/A translocation as the reference Clostridial toxin translocation domain on domain variant comprising amino acids 449-873 of SEQID which the naturally occurring Clostridial toxin translocation NO: 1 will have at least one amino acid difference, such as, domain variant is based, and can be substituted for the refer e.g., an amino acid Substitution, deletion or addition, as com ence Clostridial toxin translocation domain in any aspect of pared to the amino acid region 449-873 of SEQID NO: 1; a the present invention. A naturally occurring Clostridial toxin BoNT/B translocation domain variant comprising amino translocation domain variant may substitute one or more acids 442-860 of SEQID NO: 2 will have at least one amino amino acids, two or more amino acids, three or more amino acid difference. Such as, e.g., an amino acid substitution, acids, four or more amino acids, five or more amino acids, ten deletion or addition, as compared to the amino acid region or more amino acids, 20 or more amino acids, 30 or more 442-860 of SEQID NO: 2; a BoNT/C1 translocation domain amino acids, 40 or more amino acids, 50 or more amino acids variant comprising amino acids 450-868 of SEQ ID NO: 3 or 100 or more amino acids from the reference Clostridial will have at least one amino acid difference, such as, e.g., an toxin translocation domain on which the naturally occurring amino acid Substitution, deletion or addition, as compared to Clostridial toxin translocation domain variant is based. A the amino acid region 450-868 of SEQID NO:3: a BoNT/D naturally occurring Clostridial toxin translocation domain translocation domain variant comprising amino acids 446 variant can also substitute at least 10 contiguous amino acids, 864 of SEQ ID NO. 4 will have at least one amino acid at least 15 contiguous amino acids, at least 20 contiguous difference. Such as, e.g., an amino acid Substitution, deletion amino acids, or at least 25 contiguous amino acids from the or addition, as compared to the amino acid region 446-864 of reference Clostridial toxin translocation domain on which the SEQID NO: 4; a BoNT/E translocation domain variant com naturally occurring Clostridial toxin translocation domain prising amino acids 423-847 of SEQID NO: 5 will have at variant is based, that possess at least 50% amino acid identity, least one amino acid difference, Such as, e.g., an amino acid 65% amino acid identity, 75% amino acid identity, 85% Substitution, deletion or addition, as compared to the amino amino acid identity or 95% amino acid identity to the refer acid region 423-847 of SEQID NO: 5; a BoNT/F transloca ence Clostridial toxin translocation domain on which the tion domain variant comprising amino acids 440-866 of SEQ naturally occurring Clostridial toxin translocation domain ID NO: 6 will have at least one amino acid difference, such as, variant is based. e.g., an amino acid Substitution, deletion or addition, as com I0081. A non-limiting examples of a naturally occurring pared to the amino acid region 440-866 of SEQID NO: 6; a Clostridial toxin translocation domain variant is a Clostridial BoNT/G translocation domain variant comprising amino toxin translocation domain isoform such as, e.g., a BoNT/A US 2009/01171.57 A1 May 7, 2009
translocation domain isoform, a BoNT/B translocation more amino acids, ten or more amino acids, 20 or more amino domain isoform, a BoNT/C1 translocation domain isoform, a acids, 30 or more amino acids, 40 or more amino acids, 50 or BoNTVD translocation domain isoform, a BoNT/E transloca more amino acids, 100 or more amino acids, or 200 or more tion domain isoform, a BoNT/F translocation domain iso amino acids from the reference Clostridial toxin translocation form, a BoNT/G translocation domain isoform, a TeNT trans domain on which the conservative Clostridial toxin translo location domain isoform, a BaNT translocation domain cation domain variant is based. A conservative Clostridial isoform, and a BuNT translocation domain isoform. A toxin translocation domain variant can also Substitute at least Clostridial toxin translocation domain isoform can function 10 contiguous amino acids, at least 15 contiguous amino in substantially the same manner as the reference Clostridial acids, at least 20 contiguous amino acids, or at least 25 con toxin translocation domain on which the Clostridial toxin tiguous amino acids from the reference Clostridial toxin translocation domain isoform is based, and can be substituted translocation domain on which the conservative Clostridial for the reference Clostridial toxin translocation domain in any toxin translocation domain variant is based, that possess at aspect of the present invention. least 50% amino acid identity, 65% amino acid identity, 75% 0082 Another non-limiting examples of a naturally occur amino acid identity, 85% amino acid identity or 95% amino ring Clostridial toxin translocation domain variant is a acid identity to the reference Clostridial toxin translocation Clostridial toxin translocation domain Subtype Such as, e.g., a domain on which the conservative Clostridial toxin translo translocation domain from subtype BoNT/A1, BoNT/A2, cation domain variant is based. Non-limiting examples of a BoNT/A3, BoNT/A4, and BoNT/A5; a translocation domain conservative Clostridial toxin translocation domain variant from subtype BoNT/B1, BoNT/B2, BoNT/B bivalent and include, e.g., conservative BoNT/A translocation domain BoNT/B nonproteolytic; a translocation domain from sub variants, conservative BoNT/B translocation domain vari type BoNT/C1-1 and BoNT/C1-2; a translocation domain ants, conservative BoNT/C1 translocation domain variants, from subtype BoNT/E1, BoNT/E2 and BoNT/E3; and a conservative BoNTVD translocation domain variants, conser translocation domain from subtype BoNT/F1, BoNT/F2, vative BoNT/E translocation domain variants, conservative BoNT/F3 and BoNT/F4. A Clostridial toxin translocation BoNTVF translocation domain variants, conservative domain Subtype can function in Substantially the same man BoNT/G translocation domain variants, conservative TeNT ner as the reference Clostridial toxin translocation domain on translocation domain variants, conservative BaNT transloca which the Clostridial toxin translocation domain subtype is tion domain variants, and conservative BuNT translocation based, and can be substituted for the reference Clostridial domain variants. toxin translocation domain in any aspect of the present inven 0085. As used herein, the term “non-conservative tion. Clostridial toxin translocation domain variant’ means a 0083. As used herein, the term “non-naturally occurring Clostridial toxin translocation domain in which 1) at least one Clostridial toxin translocation domain variant’ means any amino acid is deleted from the reference Clostridial toxin Clostridial toxin translocation domain produced with the aid translocation domain on which the non-conservative of human manipulation, including, without limitation, Clostridial toxin translocation domain variant is based; 2) at Clostridial toxin translocation domains produced by genetic least one amino acid added to the reference Clostridial toxin engineering using random mutagenesis or rational design and translocation domain on which the non-conservative Clostridial toxin translocation domains produced by chemi Clostridial toxin translocation domain is based; or 3) at least cal synthesis. Non-limiting examples of non-naturally occur one amino acid is Substituted by another amino acid or an ring Clostridial toxin translocation domain variants include, amino acid analog that does not share any property similar to e.g., conservative Clostridial toxin translocation domain vari that of the original amino acid from the reference Clostridial ants, non-conservative Clostridial toxin translocation domain toxin translocation domain sequence (Table 1). A non-con variants, Clostridial toxin translocation domain chimeric servative Clostridial toxin translocation domain variant can variants and active Clostridial toxin translocation domain function in Substantially the same manner as the reference fragments. Clostridial toxin translocation domain on which the non 0084 As used herein, the term “conservative Clostridial conservative Clostridial toxin translocation domain variant is toxin translocation domain variant’ means a Clostridial toxin based, and can be substituted for the reference Clostridial translocation domain that has at least one amino acid Substi toxin translocation domain in any aspect of the present inven tuted by another amino acid or an amino acid analog that has tion. A non-conservative Clostridial toxin translocation at least one property similar to that of the original amino acid domain variant can delete one or more amino acids, two or from the reference Clostridial toxin translocation domain more amino acids, three or more amino acids, four or more sequence (Table 1). Examples of properties include, without amino acids, five or more amino acids, and ten or more amino limitation, similar size, topography, charge, hydrophobicity, acids from the reference Clostridial toxin translocation hydrophilicity, lipophilicity, covalent-bonding capacity, domain on which the non-conservative Clostridial toxin hydrogen-bonding capacity, a physicochemical property, of translocation domain variant is based. A non-conservative the like, or any combination thereof. A conservative Clostridial toxin translocation domain variant can add one or Clostridial toxin translocation domain variant can function in more amino acids, two or more amino acids, three or more substantially the same manner as the reference Clostridial amino acids, four or more amino acids, five or more amino toxin translocation domain on which the conservative acids, and tenor more amino acids to the reference Clostridial Clostridial toxin translocation domain variant is based, and toxin translocation domain on which the non-conservative can be substituted for the reference Clostridial toxin translo Clostridial toxin translocation domain variant is based. A cation domain in any aspect of the present invention. A con non-conservative Clostridial toxin translocation domain vari servative Clostridial toxin translocation domain variant may ant may substitute one or more amino acids, two or more Substitute one or more amino acids, two or more amino acids, amino acids, three or more amino acids, four or more amino three or more amino acids, four or more amino acids, five or acids, five or more amino acids, ten or more amino acids, 20 US 2009/01171.57 A1 May 7, 2009
or more amino acids, 30 or more amino acids, 40 or more I0088 Any of a variety of sequence alignment methods can amino acids, 50 or more amino acids, 100 or more amino be used to determine percent identity of naturally-occurring acids, or 200 or more amino acids from the reference Clostridial toxin translocation domain variants and non-natu Clostridial toxin translocation domain on which the non rally-occurring Clostridial toxin translocation domain vari conservative Clostridial toxin translocation domain variant is ants, including, without limitation, global methods, local based. A non-conservative Clostridial toxin translocation methods and hybrid methods, such as, e.g., segment approach domain variant can also Substitute at least 10 contiguous methods. Protocols to determine percent identity are routine amino acids, at least 15 contiguous amino acids, at least 20 procedures within the scope of one skilled in the art and from contiguous amino acids, or at least 25 contiguous amino acids the teaching herein. from the reference Clostridial toxin translocation domain on 0089. Thus, in an embodiment, a modified Clostridial which the non-conservative Clostridial toxin translocation toxin disclosed in the present specification comprises a domain variant is based, that possess at least 50% amino acid Clostridial toxin translocation domain. In an aspect of this identity, 65% amino acid identity, 75% amino acid identity, embodiment, a Clostridial toxin translocation domain com 85% amino acid identity or 95% amino acid identity to the prises a naturally occurring Clostridial toxin translocation reference Clostridial toxin translocation domain on which the domain variant, such as, e.g., a Clostridial toxin translocation non-conservative Clostridial toxin translocation domain vari domain isoform or a Clostridial toxin translocation domain ant is based. Non-limiting examples of a non-conservative subtype. In another aspect of this embodiment, a Clostridial Clostridial toxin translocation domain variant include, e.g., toxin translocation domain comprises a non-naturally occur non-conservative BoNT/A translocation domain variants, ring Clostridial toxin translocation domain variant, such as, non-conservative BoNT/B translocation domain variants, e.g., a conservative Clostridial toxin translocation domain non-conservative BoNT/C1 translocation domain variants, variant, a non-conservative Clostridial toxin translocation non-conservative BoNTVD translocation domain variants, domain variant, a Clostridial toxin chimeric translocation non-conservative BoNT/E translocation domain variants, domain, an active Clostridial toxin translocation domain frag non-conservative BoNT/F translocation domain variants, ment, or any combination thereof. non-conservative BoNT/G translocation domain variants, 0090. In another embodiment, a Clostridial toxin translo and non-conservative TeNT translocation domain variants, cation domain comprises a BoNT/A translocation domain. In non-conservative BaNT translocation domain variants, and an aspect of this embodiment, a BoNT/A translocation non-conservative BuNT translocation domain variants. domain comprises amino acids 449-873 of SEQID NO: 1. In 0.086 As used herein, the term “Clostridial toxin translo another aspect of this embodiment, a BoNT/A translocation cation domain chimeric' means a polypeptide comprising at domain comprises a naturally occurring BoNT/A transloca least a portion of a Clostridial toxin translocation domain and tion domain variant, such as, e.g., a translocation domain at least a portion of at least one other polypeptide to form a from a BoNT/A isoform or a translocation domain from a toxin translocation domain with at least one property differ BoNT/A subtype. In another aspect of this embodiment, a ent from the reference Clostridial toxin translocation domains BoNT/A translocation domain comprises amino acids 449 of Table 1, with the proviso that this Clostridial toxin trans 873 of a naturally occurring BoNT/A translocation domain location domain chimeric is still capable of specifically tar variant of SEQID NO: 1, such as, e.g., amino acids 449-873 geting the core components of the neurotransmitter release of a BoNT/A isoform of SEQ ID NO: 1 or amino acids apparatus and thus participate in executing the overall cellular 449-873 of a BoNT/A subtype of SEQ ID NO: 1. In still mechanism whereby a Clostridial toxin proteolytically another aspect of this embodiment, a BoNT/A translocation cleaves a substrate. domain comprises a non-naturally occurring BoNT/A trans 0087. As used herein, the term “active Clostridial toxin location domain variant, such as, e.g., a conservative BoNT/A translocation domain fragment’ means any of a variety of translocation domain variant, a non-conservative BoNT/A Clostridial toxin fragments comprising the translocation translocation domain variant, a BoNT/A chimeric transloca domain can be useful in aspects of the present invention with tion domain, an active BoNT/A translocation domain frag the proviso that these active fragments can facilitate the ment, or any combination thereof. In still another aspect of release of the LC from intracellular vesicles into the cyto this embodiment, a BoNT/A translocation domain comprises plasm of the target cell and thus participate in executing the amino acids 449-873 of a non-naturally occurring BoNT/A overall cellular mechanism whereby a Clostridial toxin pro translocation domain variant of SEQID NO: 1, such as, e.g., teolytically cleaves a Substrate. The translocation domains amino acids 449-873 of a conservative BoNT/A translocation from the heavy chains of Clostridial toxins are approximately domain variant of SEQID NO: 1, amino acids 449-873 of a 410-430 amino acids in length and comprise a translocation non-conservative BoNT/A translocation domain variant of domain (Table 1). Research has shown that the entire length SEQ ID NO: 1, amino acids 449-873 of an active BoNT/A ofa translocation domain from a Clostridial toxin heavy chain translocation domain fragment of SEQ ID NO: 1, or any is not necessary for the translocating activity of the translo combination thereof. cation domain. Thus, aspects of this embodiment can include (0091. In other aspects of this embodiment, a BoNT/A Clostridial toxin translocation domains comprising a translo translocation domain comprises a polypeptide having an cation domain having a length of e.g., at least 350 amino amino acid identity to amino acids 449-873 of SEQID NO: 1 acids, at least 375 amino acids, at least 400 amino acids and at of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, least 425 amino acids. Other aspects of this embodiment can at least 90% or at least 95%. In yet other aspects of this include Clostridial toxin translocation domains comprising embodiment, a BoNT/A translocation domain comprises a translocation domain having a length of, e.g., at most 350 polypeptide having an amino acid identity to amino acids amino acids, at most 375 amino acids, at most 400 amino 449-873 of SEQID NO: 1 of, e.g., at most 70%, at most 75%, acids and at most 425 amino acids. at most 80%, at most 85%, at most 90% or at most 95%. US 2009/01171.57 A1 May 7, 2009
0092. In other aspects of this embodiment, a BoNT/A (0095. In other aspects of this embodiment, a BoNT/B translocation domain comprises a polypeptide having, e.g., at translocation domain comprises a polypeptide having an most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 amino acid identity to amino acids 442-860 of SEQID NO: 2 non-contiguous amino acid substitutions relative to amino of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, acids 449-873 of SEQID NO: 1; at least 1, 2, 3, 4, 5, 6, 7, 8, at least 90% or at least 95%. In yet other aspects of this 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid embodiment, a BoNT/B translocation domain comprises a substitutions relative to amino acids 449-873 of SEQID NO: polypeptide having an amino acid identity to amino acids 1; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 442-860 of SEQID NO: 2 of, e.g., at most 70%, at most 75%, non-contiguous amino acid deletions relative to amino acids at most 80%, at most 85%, at most 90% or at most 95%. 449-873 of SEQID NO: 1; at least 1,2,3,4,5,6,7,8,9, 10, (0096. In other aspects of this embodiment, a BoNT/B 20, 30, 40, 50, or 100 non-contiguous amino acid deletions translocation domain comprises a polypeptide having, e.g., at relative to amino acids 449-873 of SEQID NO: 1; at most 1, most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous non-contiguous amino acid substitutions relative to amino acids 442-860 of SEQID NO: 2; at least 1, 2, 3, 4, 5, 6, 7, 8, amino acid additions relative to amino acids 449-873 of SEQ 9, 10, 20, 30, 40, 50, 100 or 200 non-contiguous amino acid ID NO: 1; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, substitutions relative to amino acids 442-860 of SEQID NO: or 100 non-contiguous amino acid additions relative to amino 2; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 acids 449-873 of SEQID NO: 1. non-contiguous amino acid deletions relative to amino acids 0093. In other aspects of this embodiment, a BoNT/A 442-860 of SEQID NO: 2; at least 1, 2, 3, 4, 5, 6,7,8,9, 10, translocation domain comprises a polypeptide having, e.g., at 20, 30, 40, 50, or 100 non-contiguous amino acid deletions most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 relative to amino acids 442-860 of SEQID NO: 2; at most 1, contiguous amino acid Substitutions relative to amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous 449-873 of SEQID NO: 1; at least 1,2,3,4,5,6,7,8,9, 10, amino acid additions relative to amino acids 442-860 of SEQ 20, 30, 40, 50, 100 or 200 contiguous amino acid substitutions ID NO: 2; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, relative to amino acids 449-873 of SEQID NO: 1; at most 1, or 100 non-contiguous amino acid additions relative to amino 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acids 442-860 of SEQID NO: 2. aciddeletions relative to amino acids 449-873 of SEQID NO: (0097. In other aspects of this embodiment, a BoNT/B 1; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 translocation domain comprises a polypeptide having, e.g., at contiguous amino acid deletions relative to amino acids 449 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 873 of SEQID NO: 1; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, contiguous amino acid Substitutions relative to amino acids 30, 40, 50, or 100 contiguous amino acid additions relative to 442-860 of SEQID NO: 2; at least 1, 2, 3, 4, 5, 6,7,8,9, 10, amino acids 449-873 of SEQID NO: 1; or at least 1, 2, 3, 4, 20, 30, 40, 50, or 100 contiguous amino acid substitutions 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid relative to amino acids 442-860 of SEQID NO: 2; at most 1, additions relative to amino acids 449-873 of SEQID NO: 1. 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino 0094. In another embodiment, a Clostridial toxin translo aciddeletions relative to amino acids 442-860 of SEQID NO: cation domain comprises a BoNT/B translocation domain. In 2; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 an aspect of this embodiment, a BoNT/B translocation contiguous amino acid deletions relative to amino acids 442 domain comprises amino acids 442-860 of SEQID NO: 2. In 860 of SEQID NO: 2; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, another aspect of this embodiment, a BoNT/B translocation 30, 40, 50, or 100 contiguous amino acid additions relative to domain comprises a naturally occurring BoNT/B transloca amino acids 442-860 of SEQID NO: 2; or at least 1, 2, 3, 4, tion domain variant, such as, e.g., a translocation domain 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid from a BoNT/B isoform or a translocation domain from a additions relative to amino acids 442-860 of SEQID NO: 2. BoNT/B subtype. In another aspect of this embodiment, a 0098. In another embodiment, a Clostridial toxin translo BoNT/B translocation domain comprises amino acids 442 cation domain comprises a BoNT/C1 translocation domain. 860 of a naturally occurring BoNT/B translocation domain In an aspect of this embodiment, a BoNT/C1 translocation variant of SEQID NO: 2, such as, e.g., amino acids 442-860 domain comprises amino acids 450-868 of SEQID NO:3. In of a BoNT/B isoform of SEQ ID NO: 2 or amino acids another aspect of this embodiment, a BoNT/C1 translocation 442-860 of a BoNT/B subtype of SEQ ID NO: 2. In still domain comprises a naturally occurring BoNT/C1 transloca another aspect of this embodiment, a BoNT/B translocation tion domain variant, such as, e.g., a translocation domain domain comprises a non-naturally occurring BoNT/B trans from a BoNT/C1 isoform or a translocation domain from a location domain variant, such as, e.g., a conservative BoNT/B BoNT/C1 subtype. In another aspect of this embodiment, a translocation domain variant, a non-conservative BoNT/B BoNT/C1 translocation domain comprises amino acids 450 translocation domain variant, a BoNT/B chimeric transloca 868 of a naturally occurring BoNT/C1 translocation domain tion domain, an active BoNT/B translocation domain frag variant of SEQID NO:3, such as, e.g., amino acids 450-868 ment, or any combination thereof. In still another aspect of of a BoNT/C1 isoform of SEQ ID NO: 3 or amino acids this embodiment, a BoNT/B translocation domain comprises 450-868 of a BoNT/C1 subtype of SEQ ID NO: 3. In still amino acids 442-860 of a non-naturally occurring BoNT/B another aspect of this embodiment, a BoNT/C1 translocation translocation domain variant of SEQID NO: 2, such as, e.g., domain comprises a non-naturally occurring BoNT/C1 trans amino acids 442-860 of a conservative BoNT/B translocation location domain variant, Such as, e.g., a conservative BoNT/ domain variant of SEQID NO: 2, amino acids 442-860 of a C1 translocation domain variant, a non-conservative BoNT/ non-conservative BoNT/B translocation domain variant of C1 translocation domain variant, a BoNT/C1 chimeric SEQ ID NO: 2, amino acids 442-860 of an active BoNT/B translocation domain, an active BoNT/C1 translocation translocation domain fragment of SEQ ID NO: 2, or any domain fragment, or any combination thereof. In still another combination thereof. aspect of this embodiment, a BoNT/C1 translocation domain US 2009/01171.57 A1 May 7, 2009
comprises amino acids 450-868 of a non-naturally occurring another aspect of this embodiment, a BoNTVD translocation BoNT/C1 translocation domain variant of SEQ ID NO: 3, domain comprises a non-naturally occurring BoNT/D trans such as, e.g., amino acids 450-868 of a conservative BoNT/ location domain variant, such as, e.g., a conservative BoNTVD C1 translocation domain variant of SEQ ID NO: 3, amino translocation domain variant, a non-conservative BoNTVD acids 450-868 of a non-conservative BoNT/C1 translocation translocation domain variant, a BoNTVD chimeric transloca domain variant of SEQID NO:3, amino acids 450-868 of an tion domain, an active BoNT/D translocation domain frag active BoNT/C1 translocation domain fragment of SEQ ID ment, or any combination thereof. In still another aspect of NO:3, or any combination thereof. this embodiment, a BoNT/D translocation domain comprises 0099. In other aspects of this embodiment, a BoNT/C1 amino acids 446-864 of a non-naturally occurring BoNT/D translocation domain comprises a polypeptide having an translocation domain variant of SEQID NO: 4, such as, e.g., amino acid identity to amino acids 450-868 of SEQID NO: 3 amino acids 446-864 of a conservative BoNTVD translocation of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, domain variant of SEQID NO: 4, amino acids 446-864 of a at least 90% or at least 95%. In yet other aspects of this non-conservative BoNTVD translocation domain variant of embodiment, a BoNT/C1 translocation domain comprises a SEQ ID NO: 4, amino acids 446-864 of an active BoNT/D polypeptide having an amino acid identity to amino acids translocation domain fragment of SEQ ID NO: 4, or any 450-868 of SEQID NO:3 of, e.g., at most 70%, at most 75%, combination thereof. at most 80%, at most 85%, at most 90% or at most 95%. (0103) In other aspects of this embodiment, a BoNT/D 0100. In other aspects of this embodiment, a BoNT/C1 translocation domain comprises a polypeptide having an translocation domain comprises a polypeptide having, e.g., at amino acid identity to amino acids 446-864 of SEQID NO: 4 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, non-contiguous amino acid substitutions relative to amino at least 90% or at least 95%. In yet other aspects of this acids 450-868 of SEQID NO:3: at least 1, 2, 3, 4, 5, 6, 7, 8, embodiment, a BoNT/D translocation domain comprises a 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid polypeptide having an amino acid identity to amino acids substitutions relative to amino acids 450-868 of SEQID NO: 446-864 of SEQID NO:4 of, e.g., at most 70%, at most 75%, 3; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 at most 80%, at most 85%, at most 90% or at most 95%. non-contiguous amino acid deletions relative to amino acids 0104. In other aspects of this embodiment, a BoNT/D 450-868 of SEQID NO:3: at least 1,2,3,4,5,6,7,8,9, 10, translocation domain comprises a polypeptide having, e.g., at 20, 30, 40, 50, or 100 non-contiguous amino acid deletions most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 relative to amino acids 450-868 of SEQID NO: 3; at most 1, non-contiguous amino acid substitutions relative to amino 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous acids 446-864 of SEQID NO: 4; at least 1, 2, 3, 4, 5, 6, 7, 8, amino acid additions relative to amino acids 450-868 of SEQ 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid ID NO:3: or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, substitutions relative to amino acids 446-864 of SEQID NO: or 100 non-contiguous amino acid additions relative to amino 4; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 acids 450-868 of SEQID NO:3. non-contiguous amino acid deletions relative to amino acids 0101. In other aspects of this embodiment, a BoNT/C1 446-864 of SEQID NO: 4; at least 1, 2, 3, 4, 5, 6,7,8,9, 10, translocation domain comprises a polypeptide having, e.g., at 20, 30, 40, 50, or 100 non-contiguous amino acid deletions most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 relative to amino acids 446-864 of SEQID NO: 4; at most 1, contiguous amino acid Substitutions relative to amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous 450-868 of SEQID NO:3: at least 1,2,3,4,5,6,7,8,9, 10, amino acid additions relative to amino acids 446-864 of SEQ 20, 30, 40, 50, or 100 contiguous amino acid substitutions ID NO: 4; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, relative to amino acids 450-868 of SEQID NO:3: at most 1, or 100 non-contiguous amino acid additions relative to amino 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acids 446-864 of SEQID NO: 4. aciddeletions relative to amino acids 450-868 of SEQID NO: 0105. In other aspects of this embodiment, a BoNT/D 3; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 translocation domain comprises a polypeptide having, e.g., at contiguous amino acid deletions relative to amino acids 450 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 868 of SEQID NO:3: at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, contiguous amino acid Substitutions relative to amino acids 30, 40, 50, or 100 contiguous amino acid additions relative to 446-864 of SEQID NO: 4; at least 1, 2, 3, 4, 5, 6,7,8,9, 10, amino acids 450-868 of SEQID NO:3: at least 1,2,3,4,5,6, 20, 30, 40, 50, or 100 contiguous amino acid substitutions 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid relative to amino acids 446-864 of SEQID NO: 4; at most 1, additions relative to amino acids 450-868 of SEQID NO:3. 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino 0102. In another embodiment, a Clostridial toxin translo aciddeletions relative to amino acids 446-864 of SEQID NO: cation domain comprises a BoNTVD translocation domain. In 4; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 an aspect of this embodiment, a BoNT/D translocation contiguous amino acid deletions relative to amino acids 446 domain comprises amino acids 446-864 of SEQID NO: 4. In 864 of SEQID NO: 4; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, another aspect of this embodiment, a BoNT/D translocation 30, 40, 50, or 100 contiguous amino acid additions relative to domain comprises a naturally occurring BoNT/D transloca amino acids 446-864 of SEQID NO: 4; or at least 1, 2, 3, 4, tion domain variant, such as, e.g., a translocation domain 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid from a BoNTVD isoform or a translocation domain from a additions relative to amino acids 446-864 of SEQID NO: 4. BoNT/D subtype. In another aspect of this embodiment, a 0106. In another embodiment, a Clostridial toxin translo BoNT/D translocation domain comprises amino acids 446 cation domain comprises a BoNT/E translocation domain. In 864 of a naturally occurring BoNT/D translocation domain an aspect of this embodiment, a BoNT/E translocation variant of SEQID NO: 4, such as, e.g., amino acids 446-864 domain comprises amino acids 423-847 of SEQID NO: 5. In of a BoNT/D isoform of SEQ ID NO. 4 or amino acids another aspect of this embodiment, a BoNT/E translocation 446-864 of a BoNT/D subtype of SEQ ID NO: 4. In still domain comprises a naturally occurring BoNT/E transloca US 2009/01171.57 A1 May 7, 2009
tion domain variant, such as, e.g., a translocation domain 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid from a BoNT/E isoform or a translocation domain from a additions relative to amino acids 423-847 of SEQID NO: 5. BoNT/E subtype. In another aspect of this embodiment, a 0110. In another embodiment, a Clostridial toxin translo BoNT/E translocation domain comprises amino acids 423 cation domain comprises a BoNT/F translocation domain. In 847 of a naturally occurring BoNT/E translocation domain an aspect of this embodiment, a BoNT/F translocation variant of SEQID NO: 5, such as, e.g., amino acids 423-847 domain comprises amino acids 440-866 of SEQID NO: 6. In of a BoNT/E isoform of SEQ ID NO: 5 or amino acids another aspect of this embodiment, a BoNT/F translocation 423-847 of a BoNT/E subtype of SEQ ID NO: 5. In still domain comprises a naturally occurring BoNT/F transloca another aspect of this embodiment, a BoNT/E translocation tion domain variant, such as, e.g., a translocation domain domain comprises a non-naturally occurring BoNT/E trans from a BoNT/F isoform or a translocation domain from a location domain variant, such as, e.g., a conservative BoNT/E BoNT/F subtype. In another aspect of this embodiment, a translocation domain variant, a non-conservative BoNT/E BoNT/F translocation domain comprises amino acids 440 translocation domain variant, a BoNT/E chimeric transloca 866 of a naturally occurring BoNT/F translocation domain tion domain, an active BoNT/E translocation domain frag variant of SEQID NO: 6, such as, e.g., amino acids 440-866 ment, or any combination thereof. In still another aspect of of a BoNT/F isoform of SEQ ID NO: 6 or amino acids this embodiment, a BoNT/E translocation domain comprises 440-866 of a BoNT/F subtype of SEQ ID NO: 6. In still amino acids 423-847 of a non-naturally occurring BoNT/E another aspect of this embodiment, a BoNT/F translocation translocation domain variant of SEQID NO: 5, such as, e.g., domain comprises a non-naturally occurring BoNT/F trans amino acids 423-847 of a conservative BoNT/E translocation location domain variant, Such as, e.g., a conservative BoNT/F domain variant of SEQID NO: 5, amino acids 423-847 of a translocation domain variant, a non-conservative BoNT/F non-conservative BoNT/E translocation domain variant of translocation domain variant, a BoNT/F chimeric transloca SEQ ID NO: 5, amino acids 423-847 of an active BoNT/E tion domain, an active BoNT/F translocation domain frag translocation domain fragment of SEQ ID NO: 5, or any ment, or any combination thereof. In still another aspect of combination thereof. this embodiment, a BoNT/F translocation domain comprises 0107. In other aspects of this embodiment, a BoNT/E amino acids 440-866 of a non-naturally occurring BoNT/F translocation domain comprises a polypeptide having an translocation domain variant of SEQID NO: 6, such as, e.g., amino acid identity to amino acids 423-847 of SEQID NO: 5 amino acids 440-866 of a conservative BoNTVF translocation of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, domain variant of SEQID NO: 6, amino acids 440-866 of a at least 90% or at least 95%. In yet other aspects of this non-conservative BoNTVF translocation domain variant of embodiment, a BoNT/E translocation domain comprises a SEQ ID NO: 6, amino acids 440-866 of an active BoNT/F polypeptide having an amino acid identity to amino acids translocation domain fragment of SEQ ID NO: 6, or any 423-847 of SEQID NO:5 of, e.g., at most 70%, at most 75%, combination thereof. at most 80%, at most 85%, at most 90% or at most 95%. 0111. In other aspects of this embodiment, a BoNT/F 0108. In other aspects of this embodiment, a BoNT/E translocation domain comprises a polypeptide having an translocation domain comprises a polypeptide having, e.g., at amino acid identity to amino acids 440-866 of SEQID NO: 6 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, non-contiguous amino acid substitutions relative to amino at least 90% or at least 95%. In yet other aspects of this acids 423-847 of SEQID NO: 5; at least 1, 2, 3, 4, 5, 6, 7, 8, embodiment, a BoNT/F translocation domain comprises a 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid polypeptide having an amino acid identity to amino acids substitutions relative to amino acids 423-847 of SEQID NO: 440-866 of SEQID NO: 6 of, e.g., at most 70%, at most 75%, 5; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 at most 80%, at most 85%, at most 90% or at most 95%. non-contiguous amino acid deletions relative to amino acids 0112. In other aspects of this embodiment, a BoNT/F 423-847 of SEQID NO: 5; at least 1,2,3,4,5,6,7,8,9, 10, translocation domain comprises a polypeptide having, e.g., at 20, 30, 40, 50, or 100 non-contiguous amino acid deletions most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 relative to amino acids 423-847 of SEQID NO: 5; at most 1, non-contiguous amino acid substitutions relative to amino 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous acids 440-866 of SEQID NO: 6; at least 1, 2, 3, 4, 5, 6, 7, 8, amino acid additions relative to amino acids 423-847 of SEQ 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid ID NO: 5; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, substitutions relative to amino acids 440-866 of SEQID NO: or 100 non-contiguous amino acid additions relative to amino 6; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 acids 423-847 of SEQID NO: 5. non-contiguous amino acid deletions relative to amino acids 0109. In other aspects of this embodiment, a BoNT/E 440-866 of SEQID NO: 6; at least 1, 2, 3, 4, 5, 6,7,8,9, 10, translocation domain comprises a polypeptide having, e.g., at 20, 30, 40, 50, or 100 non-contiguous amino acid deletions most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 relative to amino acids 440-866 of SEQID NO: 6; at most 1, contiguous amino acid Substitutions relative to amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous 423-847 of SEQID NO: 5; at least 1,2,3,4,5,6,7,8,9, 10, amino acid additions relative to amino acids 440-866 of SEQ 20, 30, 40, 50, or 100 contiguous amino acid substitutions ID NO: 6; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, relative to amino acids 423-847 of SEQID NO: 5; at most 1, or 100 non-contiguous amino acid additions relative to amino 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acids 440-866 of SEQID NO: 6. aciddeletions relative to amino acids 423-847 of SEQID NO: 0113. In other aspects of this embodiment, a BoNT/F 5; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 translocation domain comprises a polypeptide having, e.g., at contiguous amino acid deletions relative to amino acids 423 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 847 of SEQID NO: 5; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, contiguous amino acid Substitutions relative to amino acids 30, 40, 50, or 100 contiguous amino acid additions relative to 440-866 of SEQID NO: 6; at least 1, 2, 3, 4, 5, 6,7,8,9, 10, amino acids 423-847 of SEQID NO: 5; or at least 1, 2, 3, 4, 20, 30, 40, 50, or 100 contiguous amino acid substitutions US 2009/01171.57 A1 May 7, 2009
relative to amino acids 440-866 of SEQID NO: 6; at most 1, or 100 non-contiguous amino acid additions relative to amino 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acids 447-865 of SEQID NO: 7. aciddeletions relative to amino acids 440-866 of SEQID NO: 0116. In other aspects of this embodiment, a BoNT/G 6; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 translocation domain comprises a polypeptide having, e.g., at contiguous amino acid deletions relative to amino acids 440 most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 866 of SEQID NO: 6; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, contiguous amino acid Substitutions relative to amino acids 30, 40, 50, or 100 contiguous amino acid additions relative to 447-865 of SEQID NO: 7; at least 1, 2, 3, 4, 5, 6,7,8,9, 10, amino acids 440-866 of SEQID NO: 6; or at least 1, 2, 3, 4, 20, 30, 40, 50, or 100 contiguous amino acid substitutions 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid relative to amino acids 447-865 of SEQID NO: 7; at most 1, additions relative to amino acids 440-866 of SEQID NO: 6. 2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions relative to amino acids 447-865 of SEQID NO: 0114. In another embodiment, a Clostridial toxin translo 7; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 cation domain comprises a BoNT/G translocation domain. In contiguous amino acid deletions relative to amino acids 447 an aspect of this embodiment, a BoNT/G translocation 865 of SEQID NO: 7; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, domain comprises amino acids 447-865 of SEQID NO: 7. In 30, 40, 50, or 100 contiguous amino acid additions relative to another aspect of this embodiment, a BoNT/G translocation amino acids 447-865 of SEQID NO: 7; or at least 1, 2, 3, 4, domain comprises a naturally occurring BoNT/G transloca 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid tion domain variant, such as, e.g., a translocation domain additions relative to amino acids 447-865 of SEQID NO: 7. from a BoNT/G isoform or a translocation domain from a 0117. In another embodiment, a Clostridial toxin translo BoNT/G subtype. In another aspect of this embodiment, a cation domain comprises a TeNT translocation domain. In an BoNT/G translocation domain comprises amino acids 447 aspect of this embodiment, a TeNT translocation domain 865 of a naturally occurring BoNT/G translocation domain comprises amino acids 458-881 of SEQID NO: 8. In another variant of SEQID NO: 7, such as, e.g., amino acids 447-865 aspect of this embodiment, a TeNT translocation domain of a BoNT/G isoform of SEQ ID NO: 7 or amino acids comprises a naturally occurring TeNT translocation domain 447-865 of a BoNT/G subtype of SEQ ID NO: 7. In still variant, such as, e.g., a translocation domain from a TeNT another aspect of this embodiment, a BoNT/G translocation isoform or a translocation domain from a TeNT subtype. In domain comprises a non-naturally occurring BoNT/G trans another aspect of this embodiment, a TeNT translocation location domain variant, such as, e.g., a conservative BoNT/G domain comprises amino acids 458-881 of a naturally occur translocation domain variant, a non-conservative BoNT/G ring TeNT translocation domain variant of SEQID NO: 8, translocation domain variant, a BoNT/G chimeric transloca such as, e.g., amino acids 458-881 of a TeNT isoform of SEQ tion domain, an active BoNT/G translocation domain frag ID NO: 8 or amino acids 458-881 of a TeNT subtype of SEQ ment, or any combination thereof. In still another aspect of ID NO: 8. In still another aspect of this embodiment, a TeNT this embodiment, a BoNT/G translocation domain comprises translocation domain comprises a non-naturally occurring amino acids 447-865 of a non-naturally occurring BoNT/G TeNT translocation domain variant, Such as, e.g., a conserva translocation domain variant of SEQID NO: 7, such as, e.g., tive TeNT translocation domain variant, a non-conservative amino acids 447-865 of a conservative BoNT/G translocation TeNT translocation domain variant, a TeNT chimeric trans domain variant of SEQID NO: 7, amino acids 447-865 of a location domain, an active TeNT translocation domain frag non-conservative BoNT/G translocation domain variant of ment, or any combination thereof. In still another aspect of SEQ ID NO: 7, amino acids 447-865 of an active BoNT/G this embodiment, a TeNT translocation domain comprises translocation domain fragment of SEQ ID NO: 7, or any amino acids 458-881 of a non-naturally occurring TeNT combination thereof. In other aspects of this embodiment, a translocation domain variant of SEQID NO: 8, such as, e.g., BoNT/G translocation domain comprises a polypeptide hav amino acids 458-881 of a conservative TeNT translocation ing an amino acid identity to amino acids 447-865 of SEQID domain variant of SEQID NO: 8, amino acids 458-881 of a NO: 7 of, e.g., at least 70%, at least 75%, at least 80%, at least non-conservative TeNT translocation domain variant of SEQ 85%, at least 90% or at least 95%. In yet other aspects of this ID NO: 8, amino acids 458-881 of an active TeNT transloca embodiment, a BoNT/G translocation domain comprises a tion domain fragment of SEQID NO: 8, or any combination polypeptide having an amino acid identity to amino acids thereof. 447-865 of SEQID NO: 7 of, e.g., at most 70%, at most 75%, 0118. In other aspects of this embodiment, a TeNT trans at most 80%, at most 85%, at most 90% or at most 95%. location domain comprises a polypeptide having an amino 0115. In other aspects of this embodiment, a BoNT/G acid identity to amino acids 458-881 of SEQID NO: 8 of, e.g., translocation domain comprises a polypeptide having, e.g., at at least 70%, at least 75%, at least 80%, at least 85%, at least most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 90% or at least 95%. In yet other aspects of this embodiment, non-contiguous amino acid substitutions relative to amino a TeNT translocation domain comprises a polypeptide having acids 447-865 of SEQID NO: 7; at least 1, 2, 3, 4, 5, 6, 7, 8, an amino acid identity to amino acids 458-881 of SEQID NO: 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid 8 of, e.g., at most 70%, at most 75%, at most 80%, at most substitutions relative to amino acids 447-865 of SEQID NO: 85%, at most 90% or at most 95%. 7; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 0119. In other aspects of this embodiment, a TeNT trans non-contiguous amino acid deletions relative to amino acids location domain comprises a polypeptidehaving, e.g., at most 447-865 of SEQID NO: 7; at least 1,2,3,4,5,6,7,8,9, 10, 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous 20, 30, 40, 50, or 100 non-contiguous amino acid deletions amino acid substitutions relative to amino acids 458-881 of relative to amino acids 447-865 of SEQID NO: 7; at most 1, SEQID NO: 8; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous 50, or 100 non-contiguous amino acid substitutions relative to amino acid additions relative to amino acids 447-865 of SEQ amino acids 458-881 of SEQID NO: 8; at most 1, 2, 3, 4, 5, ID NO: 7; or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid US 2009/01171.57 A1 May 7, 2009 20 deletions relative to amino acids 458-881 of SEQID NO: 8: I0123. In other aspects of this embodiment, a BaNT trans at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 location domain comprises a polypeptidehaving, e.g., at most non-contiguous amino acid deletions relative to amino acids 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous 458-881 of SEQID NO: 8; at most 1,2,3,4,5,6,7,8,9, 10, amino acid substitutions relative to amino acids 432-857 of 20, 30, 40, 50, or 100 non-contiguous amino acid additions SEQID NO: 9; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, relative to amino acids 458-881 of SEQID NO:8; or at least 50, or 100 non-contiguous amino acid substitutions relative to 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acids 432-857 of SEQID NO: 9; at most 1, 2, 3, 4, 5, amino acid additions relative to amino acids 458-881 of SEQ 6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid ID NO: 8. deletions relative to amino acids 432-857 of SEQID NO: 9: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 0120 In other aspects of this embodiment, a TeNT trans non-contiguous amino acid deletions relative to amino acids location domain comprises a polypeptidehaving, e.g., at most 432-857 of SEQID NO: 9; at most 1,2,3,4,5,6,7,8,9, 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous 20, 30, 40, 50, or 100 non-contiguous amino acid additions amino acid substitutions relative to amino acids 458-881 of relative to amino acids 432-857 of SEQID NO:9; or at least SEQ ID NO: 8; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous 50, or 100 contiguous amino acid substitutions relative to amino acid additions relative to amino acids 432-857 of SEQ amino acids 458-881 of SEQID NO: 8; at most 1, 2, 3, 4, 5, ID NO: 9. 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid 0.124. In other aspects of this embodiment, a BaNT trans deletions relative to amino acids 458-881 of SEQID NO: 8: location domain comprises a polypeptidehaving, e.g., at most at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous contiguous amino acid deletions relative to amino acids 458 amino acid substitutions relative to amino acids 432-857 of 881 of SEQID NO: 8; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, SEQID NO: 9; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 30, 40, 50, or 100 contiguous amino acid additions relative to 50, or 100 contiguous amino acid substitutions relative to amino acids 458-881 of SEQID NO:8; or at least 1, 2, 3, 4, amino acids 432-857 of SEQID NO: 9; at most 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid additions relative to amino acids 458-881 of SEQID NO: 8. deletions relative to amino acids 432-857 of SEQID NO: 9: 0121. In another embodiment, a Clostridial toxin translo at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 cation domain comprises a BaNT translocation domain. In an contiguous amino acid deletions relative to amino acids 432 aspect of this embodiment, a BaNT translocation domain 857 of SEQID NO: 9; at most 1,2,3,4,5,6,7,8,9, 10, 20, comprises amino acids 432-857 of SEQID NO:9. In another 30, 40, 50, or 100 contiguous amino acid additions relative to aspect of this embodiment, a BaNT translocation domain amino acids 432-857 of SEQID NO:9; or at least 1, 2, 3, 4, comprises a naturally occurring BaNT translocation domain 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid variant, Such as, e.g., a translocation domain from a BaNT additions relative to amino acids 432-857 of SEQID NO: 9. isoform or a translocation domain from a BaNT subtype. In 0.125. In another embodiment, a Clostridial toxin translo another aspect of this embodiment, a BaNT translocation cation domain comprises a BuNT translocation domain. In an domain comprises amino acids 432-857 of a naturally occur aspect of this embodiment, a BuNT translocation domain ring BaNT translocation domain variant of SEQID NO: 9. comprises amino acids 423-847 of SEQ ID NO: 10. In such as, e.g., amino acids 432-857 of a BaNT isoform of SEQ another aspect of this embodiment, a BuNT translocation ID NO:9 or amino acids 432-857 of a BaNT subtype of SEQ domain comprises a naturally occurring BuNT translocation ID NO:9. In still another aspect of this embodiment, a BaNT domain variant, such as, e.g., a translocation domain from a translocation domain comprises a non-naturally occurring BuNT isoform or a translocation domain from a BuNT Sub BaNT translocation domain variant, Such as, e.g., a conser type. In another aspect of this embodiment, a BuNT translo Vative BaNT translocation domain variant, a non-conserva cation domain comprises amino acids 423-847 of a naturally tive BaNT translocation domain variant, a BaNT chimeric occurring BuNT translocation domain variant of SEQID NO: translocation domain, an active BaNT translocation domain 10, such as, e.g., amino acids 423-847 of a BuNT isoform of fragment, or any combination thereof. In still another aspect SEQ ID NO: 10 or amino acids 423-847 of a BuNT subtype of this embodiment, a BaNT translocation domain comprises of SEQID NO: 10. In still another aspect of this embodiment, amino acids 432-857 of a non-naturally occurring BaNT a BuNT translocation domain comprises a non-naturally translocation domain variant of SEQID NO: 9, such as, e.g., occurring BuNT translocation domain variant, Such as, e.g., a amino acids 432-857 of a conservative BaNT translocation conservative BuNT translocation domain variant, a non-con domain variant of SEQID NO: 9, amino acids 432-857 of a servative BuNT translocation domain variant, a BuNT chi non-conservative BaNT translocation domain variant of SEQ meric translocation domain, an active BuNT translocation ID NO: 9, amino acids 432-857 of an active BaNT transloca domain fragment, or any combination thereof. In still another tion domain fragment of SEQID NO:9, or any combination aspect of this embodiment, a BuNT translocation domain thereof. comprises amino acids 423-847 of a non-naturally occurring 0122. In other aspects of this embodiment, a BaNT trans BuNT translocation domain variant of SEQID NO: 10, such location domain comprises a polypeptide having an amino as, e.g., amino acids 423-847 of a conservative BuNT trans acid identity to amino acids 432-857 of SEQID NO:9 of, e.g., location domain variant of SEQ ID NO: 10, amino acids at least 70%, at least 75%, at least 80%, at least 85%, at least 423-847 of a non-conservative BuNT translocation domain 90% or at least 95%. In yet other aspects of this embodiment, variant of SEQID NO: 10, amino acids 423-847 of an active a BaNT translocation domain comprises a polypeptide hav BuNT translocation domain fragment of SEQID NO: 10, or ing an amino acid identity to amino acids 432-857 of SEQID any combination thereof. NO: 9 of, e.g., at most 70%, at most 75%, at most 80%, at I0126. In other aspects of this embodiment, a BuNT trans most 85%, at most 90% or at most 95%. location domain comprises a polypeptide having an amino US 2009/01171.57 A1 May 7, 2009
acid identity to amino acids 423-847 of SEQID NO: 10 of 2007); and Steward, L. E. et al., Modified Clostridial Toxins e.g., at least 70%, at least 75%, at least 80%, at least 85%, at with Enhanced Translocation Capabilities and Altered Tar least 90% or at least 95%. In yet other aspects of this embodi geting Activity For Non-Clostridial Toxin Target Cells, U.S. ment, a BuNT translocation domain comprises a polypeptide patent application Ser. No. 1 1/776,075 (Jul. 11, 2007), each having an amino acid identity to amino acids 423-847 of SEQ of which is incorporated by reference in its entirety. ID NO: 10 of, e.g., at most 70%, at most 75%, at most 80%, 0.130. A non-limiting example of an opioid peptide bind at most 85%, at most 90% or at most 95%. ing domain disclosed in the present specification is, e.g., an 0127. In other aspects of this embodiment, a BuNT trans enkephalin, an endomorphin, an endorphin, a dynorphin, a location domain comprises a polypeptidehaving, e.g., at most nociceptin or a hemorphin. Thus, in an embodiment, a bind 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous ing domain comprises an opioid peptide. amino acid substitutions relative to amino acids 423-847 of I0131. In another embodiment, an opioid peptide com SEQID NO: 10; at least 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, prises an enkephalin peptide. In aspects of this embodiment, 50, or 100 non-contiguous amino acid substitutions relative to a enkephalin peptide comprises a Leu-enkephalin, a Met amino acids 423-847 of SEQID NO: 10; at most 1, 2, 3, 4, 5, enkephalin, a Met-enkephalin MRGL or a Met-enkephalin 6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid MRF. In other aspects of this embodiment, an enkephalin deletions relative to amino acids 423-847 of SEQID NO: 10; peptide comprises SEQID NO: 52, SEQID NO:53, SEQID at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 NO:54 or SEQID NO:55. non-contiguous amino acid deletions relative to amino acids 0.132. In other aspects of this embodiment, an enkephalin 423-847 of SEQID NO: 10; at most 1,2,3,4,5,6,7,8,9, 10, comprises a polypeptide having an amino acid identity to 20, 30, 40, 50, or 100 non-contiguous amino acid additions SEQID NO:52, SEQID NO:53, SEQID NO:54 or SEQID relative to amino acids 423-847 of SEQID NO: 10; or at least NO: 55 of, e.g., at least 70%, at least 75%, at least 80%, at 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, or 100 non-contiguous least 85%, at least 90% or at least 95%. In yet other aspects of amino acid additions relative to amino acids 423-847 of SEQ this embodiment, an enkephalin comprises a polypeptide ID NO: 1.O. having an amino acid identity to SEQ ID NO: 52, SEQ ID 0128. In other aspects of this embodiment, a BuNT trans NO:53, SEQID NO:54 or SEQID NO: 55 of, e.g., at most location domain comprises a polypeptidehaving, e.g., at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous at most 95%. amino acid substitutions relative to amino acids 423-847 of I0133. In other aspects of this embodiment, an enkephalin SEQID NO: 10; at least 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, comprises a polypeptide having, e.g., at least 1, 2, or 3 non 50, or 100 contiguous amino acid substitutions relative to contiguous amino acid substitutions relative to SEQID NO: amino acids 423-847 of SEQID NO: 10; at most 1, 2, 3, 4, 5, 52, SEQID NO:53, SEQ ID NO:54 or SEQID NO. 55; at 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid most 1, 2, or 3 non-contiguous amino acid substitutions rela deletions relative to amino acids 423-847 of SEQID NO: 10; tive to SEQID NO: 52, SEQID NO:53, SEQID NO: 54 or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 SEQID NO. 55; at least 1, 2, or 3 non-contiguous amino acid contiguous amino acid deletions relative to amino acids 423 deletions relative to SEQID NO:52, SEQID NO:53, SEQID 847 of SEQID NO: 10; at most 1,2,3,4,5,6,7,8,9, 10, 20, NO: 54 or SEQID NO. 55; at most 1, 2, or 3 non-contiguous 30, 40, 50, or 100 contiguous amino acid additions relative to amino aciddeletions relative to SEQID NO:52, SEQID NO: amino acids 423-847 of SEQID NO: 10; or at least 1, 2, 3, 4, 53, SEQ ID NO. 54 or SEQ ID NO. 55; at least 1, 2, or 3 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid non-contiguous amino acid additions relative to SEQID NO: additions relative to amino acids 423-847 of SEQID NO: 10. 52, SEQID NO:53, SEQID NO:54 or SEQID NO:55; or at 0129. In another aspect of the invention, a modified most 1, 2, or 3 non-contiguous amino acid additions relative Clostridial toxin comprises, in part, an opioid peptide binding to SEQID NO:52, SEQID NO:53, SEQID NO:54 or SEQ domain. By “binding domain is meant an amino acid ID NO: 55. sequence region able to preferentially bind to a cell Surface I0134. In other aspects of this embodiment, an enkephalin marker characteristic of the target cell under physiological comprises a polypeptide having, e.g., at least 1, 2, or 3 con conditions. The cell Surface marker may comprise a polypep tiguous amino acid substitutions relative to SEQID NO: 52, tide, a polysaccharide, a lipid, a glycoprotein, a lipoprotein, or SEQID NO:53, SEQID NO: 54 or SEQID NO. 55; at most may have structural characteristics of more than one of these. 1, 2, or 3 contiguous amino acid substitutions relative to SEQ By "preferentially interact' is meant that the disassociation ID NO:52, SEQID NO:53, SEQID NO:54 or SEQID NO: constant (K) of the binding domain for the cell Surface 55; at least 1, 2, or 3 contiguous amino acid deletions relative marker is at least one order of magnitude less than that of the to SEQID NO:52, SEQID NO:53, SEQID NO:54 or SEQ binding domain for any other cell surface marker. Preferably, ID NO. 55; at most 1, 2, or 3 contiguous amino acid deletions the disassociation constant is at least 2 orders of magnitude relative to SEQID NO:52, SEQID NO:53, SEQID NO: 54 less, even more preferably the disassociation constant is at or SEQID NO. 55; at least 1, 2, or 3 contiguous amino acid least 3 orders of magnitude less than that of the binding additions relative to SEQID NO:52, SEQID NO:53, SEQID domain for any other cell surface marker to which the neuro NO: 54 or SEQID NO. 55; or at most 1, 2, or 3 contiguous toxin or modified neurotoxin is exposed. Examples of binding amino acid additions relative to SEQID NO:52, SEQID NO: domains are described in, e.g., Steward, L. E. et al., Modified 53, SEQID NO:54 or SEQID NO:55. Clostridial Toxins with Enhanced Translocation Capability I0135) In another embodiment, an opioid peptide com and Enhanced Targeting Activity, U.S. patent application Ser. prises a bovine adrenomedullary-22 (BAM22) peptide. In No. 1 1/776,043 (Jul. 11, 2007); Steward, L. E. et al., Modified aspects of this embodiment, a BAM22 peptide comprises a Clostridial Toxins with Enhanced Translocation Capabilities BAM22 peptide (1-12), a BAM22 peptide (6-22), a BAM22 and Altered Targeting Activity For Clostridial Toxin Target peptide (8-22) or a BAM22 peptide (1-22). In other aspects of Cells, U.S. patent application Ser. No. 1 1/776,052 (Jul 11, this embodiment, a BAM22 peptide comprises amino acids US 2009/01171.57 A1 May 7, 2009 22
1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 amino acids 1-22 of SEQID NO:57; amino acids 1-12, amino of SEQ ID NO: 56; amino acids 1-12, amino acids 6-22, acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 57; NO: 58; amino acids 1-12, amino acids 6-22, amino acids amino acids 1-12, amino acids 6-22, amino acids 8-22 or 8-22 or amino acids 1-22 of SEQ ID NO. 59; amino acids amino acids 1-22 of SEQID NO:58; amino acids 1-12, amino 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID of SEQ ID NO: 60; or amino acids 1-12, amino acids 6-22, NO: 59; amino acids 1-12, amino acids 6-22, amino acids amino acids 8-22 or amino acids 1-22 of SEQID NO: 61; at 8-22 or amino acids 1-22 of SEQID NO: 60 or amino acids most 1, 2, 3, 4, or 5 non-contiguous amino acid deletions 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 relative to amino acids 1-12, amino acids 6-22, amino acids of SEQID NO: 61. 8-22 or amino acids 1-22 of SEQ ID NO: 56; amino acids 0136. In other aspects of this embodiment, a BAM22 com 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 prises a polypeptide having an amino acid identity to amino of SEQ ID NO: 57; amino acids 1-12, amino acids 6-22, acids 1-12, amino acids 6-22, amino acids 8-22 or amino amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 58: acids 1-22 of SEQID NO:56; amino acids 1-12, amino acids amino acids 1-12, amino acids 6-22, amino acids 8-22 or 6-22, amino acids 8-22 or amino acids 1-22 of SEQID NO: amino acids 1-22 of SEQID NO:59; amino acids 1-12, amino 57; amino acids 1-12, amino acids 6-22, amino acids 8-22 or acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID amino acids 1-22 of SEQID NO:58; amino acids 1-12, amino NO: 60; or amino acids 1-12, amino acids 6-22, amino acids acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID 8-22 or amino acids 1-22 of SEQID NO: 61; at least 1, 2, 3, NO: 59; amino acids 1-12, amino acids 6-22, amino acids 4, or 5 non-contiguous amino acid additions relative to amino 8-22 or amino acids 1-22 of SEQID NO: 60; or amino acids acids 1-12, amino acids 6-22, amino acids 8-22 or amino 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 acids 1-22 of SEQID NO:56; amino acids 1-12, amino acids of SEQID NO: 61 of, e.g., at least 70%, at least 75%, at least 6-22, amino acids 8-22 or amino acids 1-22 of SEQID NO: 80%, at least 85%, at least 90% or at least 95%. In yet other 57; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aspects of this embodiment, a BAM22 peptide binding amino acids 1-22 of SEQID NO:58; amino acids 1-12, amino domain comprises a polypeptide having an amino acid iden acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID tity to amino acids 1-12, amino acids 6-22, amino acids 8-22 NO: 59; amino acids 1-12, amino acids 6-22, amino acids or amino acids 1-22 of SEQ ID NO: 56; amino acids 1-12, 8-22 or amino acids 1-22 of SEQID NO: 60; or amino acids amino acids 6-22, amino acids 8-22 or amino acids 1-22 of 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 SEQID NO: 57; amino acids 1-12, amino acids 6-22, amino of SEQID NO: 61; or at most 1, 2, 3, 4, or 5 non-contiguous acids 8-22 or amino acids 1-22 of SEQ ID NO: 58; amino amino acid additions relative to amino acids 1-12, amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID acids 1-22 of SEQID NO. 59; amino acids 1-12, amino acids NO: 56; amino acids 1-12, amino acids 6-22, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID NO: 8-22 or amino acids 1-22 of SEQ ID NO: 57; amino acids 60; or amino acids 1-12, amino acids 6-22, amino acids 8-22 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 or amino acids 1-22 of SEQID NO: 61 of, e.g., at most 70%, of SEQ ID NO: 58; amino acids 1-12, amino acids 6-22, at most 75%, at most 80%, at most 85%, at most 90% or at amino acids 8-22 or amino acids 1-22 of SEQ ID NO. 59: most 95%. amino acids 1-12, amino acids 6-22, amino acids 8-22 or 0.137 In other aspects of this embodiment, a BAM22 pep amino acids 1-22 of SEQID NO: 60; or amino acids 1-12, tide comprises a polypeptide having, e.g., at least 1, 2, 3, 4, or amino acids 6-22, amino acids 8-22 or amino acids 1-22 of 5 non-contiguous amino acid substitutions relative to amino SEQID NO: 61. acids 1-12, amino acids 6-22, amino acids 8-22 or amino 0.138. In other aspects of this embodiment, a BAM22 com acids 1-22 of SEQID NO:56; amino acids 1-12, amino acids prises a polypeptide having, e.g., at least 1, 2, 3, 4, or 5 6-22, amino acids 8-22 or amino acids 1-22 of SEQID NO: contiguous amino acid Substitutions relative to amino acids 57; amino acids 1-12, amino acids 6-22, amino acids 8-22 or 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 amino acids 1-22 of SEQID NO:58; amino acids 1-12, amino of SEQ ID NO: 56; amino acids 1-12, amino acids 6-22, acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 57; NO: 59; amino acids 1-12, amino acids 6-22, amino acids amino acids 1-12, amino acids 6-22, amino acids 8-22 or 8-22 or amino acids 1-22 of SEQID NO: 60; or amino acids amino acids 1-22 of SEQID NO:58; amino acids 1-12, amino 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID of SEQ ID NO: 61; at most 1, 2, 3, 4, or 5 non-contiguous NO: 59; amino acids 1-12, amino acids 6-22, amino acids amino acid Substitutions relative to amino acids 1-12, amino 8-22 or amino acids 1-22 of SEQID NO: 60; or amino acids acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 NO: 56; amino acids 1-12, amino acids 6-22, amino acids of SEQID NO: 61; at most 1, 2, 3, 4, or 5 contiguous amino 8-22 or amino acids 1-22 of SEQ ID NO: 57; amino acids acid Substitutions relative to amino acids 1-12, amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 6-22, amino acids 8-22 or amino acids 1-22 of SEQID NO: of SEQ ID NO: 58; amino acids 1-12, amino acids 6-22, 56; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 8-22 or amino acids 1-22 of SEQ ID NO. 59: amino acids 1-22 of SEQID NO:57; amino acids 1-12, amino amino acids 1-12, amino acids 6-22, amino acids 8-22 or acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID amino acids 1-22 of SEQID NO: 60; or amino acids 1-12, NO: 58; amino acids 1-12, amino acids 6-22, amino acids amino acids 6-22, amino acids 8-22 or amino acids 1-22 of 8-22 or amino acids 1-22 of SEQ ID NO. 59; amino acids SEQID NO: 61; at least 1, 2, 3, 4, or 5 non-contiguous amino 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 acid deletions relative to amino acids 1-12, amino acids 6-22, of SEQ ID NO: 60; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 56; amino acids 8-22 or amino acids 1-22 of SEQID NO: 61; at amino acids 1-12, amino acids 6-22, amino acids 8-22 or least 1, 2, 3, 4, or 5 contiguous amino acid deletions relative US 2009/01171.57 A1 May 7, 2009
to amino acids 1-12, amino acids 6-22, amino acids 8-22 or most 1, 2, or 3 non-contiguous amino acid deletions relative amino acids 1-22 of SEQID NO:56; amino acids 1-12, amino to SEQ ID NO: 62 or SEQ ID NO: 63; at least 1, 2, or 3 acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID non-contiguous amino acid additions relative to SEQID NO: NO: 57; amino acids 1-12, amino acids 6-22, amino acids 62 or SEQID NO: 63; or at most 1, 2, or 3 non-contiguous 8-22 or amino acids 1-22 of SEQ ID NO: 58; amino acids amino acid additions relative to SEQID NO: 62 or SEQID 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 NO: 63. of SEQ ID NO: 59; amino acids 1-12, amino acids 6-22, 0142. In other aspects of this embodiment, an endomor amino acids 8-22 or amino acids 1-22 of SEQID NO: 60; or phin comprises a polypeptide having, e.g., at least 1, 2, or 3 amino acids 1-12, amino acids 6-22, amino acids 8-22 or contiguous amino acid substitutions relative to SEQID NO: amino acids 1-22 of SEQID NO: 61; at most 1, 2, 3, 4, or 5 62 or SEQID NO: 63; at most 1, 2, or 3 contiguous amino acid contiguous amino acid deletions relative to amino acids 1-12, substitutions relative to SEQIDNO: 62 or SEQID NO: 63; at amino acids 6-22, amino acids 8-22 or amino acids 1-22 of least 1, 2, or 3 contiguous amino acid deletions relative to SEQID NO:56; amino acids 1-12, amino acids 6-22, amino SEQ ID NO: 62 or SEQ ID NO: 63; at most 1, 2, or 3 acids 8-22 or amino acids 1-22 of SEQ ID NO: 57; amino contiguous amino acid deletions relative to SEQID NO: 62 or acids 1-12, amino acids 6-22, amino acids 8-22 or amino SEQ ID NO: 63; at least 1, 2, or 3 contiguous amino acid acids 1-22 of SEQID NO: 58; amino acids 1-12, amino acids additions relative to SEQID NO: 62 or SEQID NO: 63; or at 6-22, amino acids 8-22 or amino acids 1-22 of SEQID NO: most 1, 2, or 3 contiguous amino acid additions relative to 59; amino acids 1-12, amino acids 6-22, amino acids 8-22 or SEQID NO: 62 or SEQID NO: 63. amino acids 1-22 of SEQID NO: 60; or amino acids 1-12, 0143. In another embodiment, an opioid peptide com amino acids 6-22, amino acids 8-22 or amino acids 1-22 of prises an endorphin peptide. In aspects of this embodiment, SEQID NO: 61; at least 1, 2, 3, 4, or 5 contiguous amino acid an endorphin peptide comprises an endorphin-C, a neoendor additions relative to amino acids 1-12, amino acids 6-22, phin-C., an endorphin-B, a neoendorphin-for an endorphin-Y. amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 56; In other aspects of this embodiment, an endorphin peptide amino acids 1-12, amino acids 6-22, amino acids 8-22 or comprises SEQID NO: 64, SEQID NO:65, SEQID NO: 66, amino acids 1-22 of SEQID NO:57; amino acids 1-12, amino SEQID NO: 67, SEQID NO: 68 or SEQ ID NO: 69. acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID 0144. In other aspects of this embodiment, an endorphin NO: 58; amino acids 1-12, amino acids 6-22, amino acids comprises a polypeptide having an amino acid identity to 8-22 or amino acids 1-22 of SEQ ID NO. 59; amino acids SEQID NO: 64, SEQ ID NO: 65, SEQID NO: 66, SEQ ID 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 NO: 67, SEQID NO: 68 or SEQID NO: 69 of, e.g., at least of SEQID NO: 60; or amino acids 1-12, amino acids 6-22, 70%, at least 75%, at least 80%, at least 85%, at least 90% or amino acids 8-22 or amino acids 1-22 of SEQID NO: 61; or at least 95%. In yet other aspects of this embodiment, an at most 1,2,3,4, or 5 contiguous amino acid additions relative endorphin comprises a polypeptide having an amino acid to amino acids 1-12, amino acids 6-22, amino acids 8-22 or identity to SEQID NO: 64, SEQID NO: 65, SEQID NO: 66, amino acids 1-22 of SEQID NO:56; amino acids 1-12, amino SEQID NO: 67, SEQID NO: 68 or SEQID NO: 69 of, e.g., acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQID at most 70%, at most 75%, at most 80%, at most 85%, at most NO: 57; amino acids 1-12, amino acids 6-22, amino acids 90% or at most 95%. 8-22 or amino acids 1-22 of SEQ ID NO: 58; amino acids 0145. In other aspects of this embodiment, an endorphin 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 comprises a polypeptide having, e.g., at least 1, 2, 3, 4, or 5 of SEQ ID NO: 59; amino acids 1-12, amino acids 6-22, non-contiguous amino acid substitutions relative to SEQID amino acids 8-22 or amino acids 1-22 of SEQID NO: 60; or NO: 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, amino acids 1-12, amino acids 6-22, amino acids 8-22 or SEQID NO: 68 or SEQID NO: 69; at most 1, 2, 3, 4, or 5 amino acids 1-22 of SEQID NO: 61. non-contiguous amino acid substitutions relative to SEQID 0.139. In another embodiment, an opioid peptide com NO: 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, prises an endomorphin peptide. In aspects of this embodi SEQID NO: 68 or SEQ ID NO: 69; at least 1, 2, 3, 4, or 5 ment, an endomorphin peptide comprises an endomorphin-1 non-contiguous amino acid deletions relative to SEQID NO: or an endomorphin-2. In other aspects of this embodiment, an 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, SEQ endomorphin peptide comprises SEQID NO: 62 or SEQID ID NO: 68 or SEQ ID NO: 69; at most 1, 2, 3, 4, or 5 NO: 63. non-contiguous amino acid deletions relative to SEQID NO: 0140. In other aspects of this embodiment, an endomor 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, SEQ phincomprises a polypeptidehaving anamino acid identity to ID NO: 68 or SEQ ID NO: 69; at least 1, 2, 3, 4, or 5 SEQ ID NO: 62 or SEQID NO: 63 of, e.g., at least 70%, at non-contiguous amino acid additions relative to SEQID NO: least 75%, at least 80%, at least 85%, at least 90% or at least 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, SEQ 95%. In yet other aspects of this embodiment, an endomor ID NO: 68 or SEQ ID NO: 69; or at most 1, 2, 3, 4, or 5 phincomprises a polypeptidehaving anamino acid identity to non-contiguous amino acid additions relative to SEQID NO: SEQ ID NO: 62 or SEQID NO: 63 of, e.g., at most 70%, at 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, SEQ most 75%, at most 80%, at most 85%, at most 90% or at most ID NO: 68 or SEQID NO: 69. 95%. 0146 In other aspects of this embodiment, an endorphin 0141. In other aspects of this embodiment, an endomor comprises a polypeptide having, e.g., at least 1, 2, 3, 4, or 5 phin comprises a polypeptide having, e.g., at least 1, 2, or 3 contiguous amino acid substitutions relative to SEQID NO: non-contiguous amino acid substitutions relative to SEQID 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, SEQ NO: 62 or SEQID NO: 63; at most 1, 2, or 3 non-contiguous ID NO: 68 or SEQ ID NO: 69; at most 1, 2, 3, 4, or 5 amino acid substitutions relative to SEQID NO: 62 or SEQ contiguous amino acid substitutions relative to SEQID NO: ID NO: 63; at least 1, 2, or 3 non-contiguous amino acid 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 67, SEQ deletions relative to SEQ ID NO: 62 or SEQ ID NO: 63; at ID NO: 68 or SEQ ID NO: 69; at least 1, 2, 3, 4, or 5 US 2009/01171.57 A1 May 7, 2009 24 contiguous amino acid deletions relative to SEQID NO: 64, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid additions SEQID NO: 65, SEQ ID NO: 66, SEQID NO: 67, SEQ ID relative to SEQID NO: 70, SEQID NO: 79 or SEQID NO: NO: 68 or SEQID NO: 69; at most 1, 2, 3, 4, or 5 contiguous 95. amino aciddeletions relative to SEQID NO: 64, SEQID NO: 0151. In another embodiment, an opioid peptide com 65, SEQID NO: 66, SEQID NO: 67, SEQID NO: 68 or SEQ prises a nociceptin peptide. In aspects of this embodiment, a ID NO: 69; at least 1, 2, 3, 4, or 5 contiguous amino acid nociceptin peptide comprises a nociceptin RK, a nociceptin, additions relative to SEQIDNO: 64, SEQID NO: 65, SEQID a neuropeptide 1, a neuropeptide 2 or a neuropeptide 3. In NO: 66, SEQID NO: 67, SEQID NO: 68 or SEQID NO: 69; other aspects of this embodiment, a nociceptin peptide com or at most 1, 2, 3, 4, or 5 contiguous amino acid additions prises SEQID NO: 101, SEQID NO: 102, SEQID NO: 103, relative to SEQID NO: 64, SEQID NO: 65, SEQID NO: 66, SEQID NO: 104, SEQID NO: 105, SEQID NO: 106, SEQ ID NO: 107, SEQID NO: 108, SEQID NO: 109 or SEQID SEQID NO: 67, SEQID NO: 68 or SEQ ID NO: 69. NO: 110. 0147 In another embodiment, an opioid peptide com 0152. In other aspects of this embodiment, a nociceptin prises a dynorphin peptide. In aspects of this embodiment, a comprises a polypeptide having an amino acid identity to dynorphin peptide comprises a dynorphin A, a dynorphin B SEQID NO: 101, SEQID NO: 108, SEQID NO: 109 or SEQ (leumorphin) or a rimorphin. In other aspects of this embodi ID NO: 110 of, e.g., at least 70%, at least 75%, at least 80%, ment, a dynorphin peptide comprises SEQID NO: 70, SEQ at least 85%, at least 90% or at least 95%. In yet other aspects IDNO: 71, SEQID NO:72, SEQIDNO:73, SEQID NO:74, of this embodiment, a nociceptin comprises a polypeptide SEQID NO: 75, SEQ ID NO: 76, SEQID NO: 77, SEQ ID having an amino acid identity to SEQID NO: 101, SEQ ID NO: 78, SEQID NO: 79, SEQID NO:80, SEQID NO: 81, NO: 108, SEQID NO: 109 or SEQ ID NO: 110 of, e.g., at SEQID NO: 82, SEQ ID NO: 83, SEQID NO: 84, SEQ ID most 70%, at most 75%, at most 80%, at most 85%, at most NO: 85, SEQID NO: 86, SEQID NO: 87, SEQID NO: 88, 90% or at most 95%. SEQID NO: 89, SEQ ID NO:90, SEQID NO: 91, SEQ ID 0153. In other aspects of this embodiment, a nociceptin NO: 92, SEQID NO: 93, SEQID NO: 94, SEQID NO:95, comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, SEQID NO: 96, SEQ ID NO: 97, SEQID NO: 98, SEQ ID 8,9, or 10 non-contiguous amino acid Substitutions relative to NO: 99 or SEQID NO: 100. SEQID NO: 101, SEQID NO: 108, SEQID NO: 109 or SEQ 0148. In other aspects of this embodiment, a dynorphin ID NO: 110; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non comprises a polypeptide having an amino acid identity to contiguous amino acid substitutions relative to SEQID NO: SEQID NO: 70, SEQID NO: 79 or SEQID NO: 95 of, e.g., 101, SEQID NO: 108, SEQID NO: 109 or SEQID NO: 110; at least 70%, at least 75%, at least 80%, at least 85%, at least at least 1,2,3,4,5,6,7,8,9, or 10 non-contiguous amino acid 90% or at least 95%. In yet other aspects of this embodiment, deletions relative to SEQID NO: 101, SEQID NO: 108, SEQ a dynorphin comprises a polypeptide having an amino acid ID NO: 109 or SEQID NO: 110; at most 1, 2, 3, 4, 5, 6, 7, 8, identity to SEQID NO: 70, SEQID NO: 79 or SEQID NO: 9, or 10 non-contiguous amino acid deletions relative to SEQ 95 of, e.g., at most 70%, at most 75%, at most 80%, at most ID NO: 101, SEQID NO: 108, SEQID NO: 109 or SEQID 85%, at most 90% or at most 95%. NO: 110; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous 0149. In other aspects of this embodiment, a dynorphin amino acid additions relative to SEQ ID NO: 101, SEQ ID comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, NO: 108, SEQID NO: 109 or SEQID NO: 110; or at most 1, 8,9, or 10 non-contiguous amino acid Substitutions relative to 2,3,4,5,6,7,8,9, or 10 non-contiguous amino acid additions SEQID NO: 70, SEQID NO: 79 or SEQID NO: 95; at most relative to SEQID NO: 101, SEQID NO: 108, SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid 109 or SEQID NO: 110. substitutions relative to SEQID NO: 70, SEQID NO: 79 or 0154) In other aspects of this embodiment, a nociceptin SEQ ID NO: 95; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, non-contiguous amino acid deletions relative to SEQID NO: 8, 9, or 10 contiguous amino acid substitutions relative to 70, SEQID NO: 79 or SEQID NO: 95; at most 1, 2, 3, 4, 5, SEQID NO: 101, SEQID NO: 108, SEQID NO: 109 or SEQ 6, 7, 8, 9, or 10 non-contiguous amino acid deletions relative ID NO: 110; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous to SEQID NO: 70, SEQID NO: 79 or SEQID NO: 95; at least amino acid substitutions relative to SEQID NO: 101, SEQID 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid NO: 108, SEQID NO: 109 or SEQID NO: 110; at least 1, 2, additions relative to SEQID NO: 70, SEQID NO: 79 or SEQ 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid deletions ID NO: 95; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non relative to SEQID NO: 101, SEQID NO: 108, SEQID NO: contiguous amino acid additions relative to SEQID NO: 70, 109 or SEQID NO: 110; at most 1,2,3,4,5,6,7,8,9, or 10 SEQID NO: 79 or SEQID NO: 95. contiguous amino acid deletions relative to SEQID NO: 101, 0150. In other aspects of this embodiment, a dynorphin SEQID NO: 108, SEQ ID NO: 109 or SEQID NO: 110; at comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid 8, 9, or 10 contiguous amino acid substitutions relative to additions relative to SEQID NO: 101, SEQID NO: 108, SEQ SEQID NO: 70, SEQID NO: 79 or SEQID NO: 95; at most ID NO: 109 or SEQID NO: 110; or at most 1, 2,3,4, 5, 6, 7, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid substitu 8,9, or 10 contiguous amino acid additions relative to SEQID tions relative to SEQID NO: 70, SEQID NO: 79 or SEQID NO: 101, SEQID NO: 108, SEQID NO: 109 or SEQID NO: NO: 95; at least 1, 2,3,4,5,6,7,8,9, or 10 contiguous amino 110. acid deletions relative to SEQID NO: 70, SEQID NO: 79 or 0155 Clostridial toxins are each translated as a single SEQ ID NO: 95; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 chain polypeptide of approximately 150 kDa that is subse contiguous amino acid deletions relative to SEQID NO: 70, quently cleaved by proteolytic Scission withina disulfide loop SEQID NO: 79 or SEQID NO: 95; at least 1,2,3,4,5,6,7, by a naturally-occurring protease (FIG. 18). This cleavage 8,9, or 10 contiguous amino acid additions relative to SEQID occurs within the discrete di-chain loop region created NO: 70, SEQID NO: 79 or SEQID NO: 95; or at most 1, 2, between two cysteine residues that form a disulfide bridge. US 2009/01171.57 A1 May 7, 2009 25
This posttranslational processing yields a di-chain molecule Clostridial toxin di-chain loop region, include, a di-chain comprising an approximately 50 kDa light chain (LC) and an loop region of BoNT/A comprising amino acids 430-454 of approximately 100 kDa heavy chain (HC) held together by SEQID NO: 1; a di-chain loop region of BoNT/B comprising the single disulfide bond and non-covalent interactions amino acids 437-446 of SEQID NO: 2; a di-chain loop region between the two chains (FIG. 2). To facilitate recombinant of BoNT/C1 comprising amino acids 437-453 of SEQ ID production of a modified Clostridial toxin, an exogenous NO:3: a di-chain loop region of BoNT/D comprising amino protease cleavage site can be used to convert the single-chain acids 437-450 of SEQ ID NO: 4; a di-chain loop region of polypeptide form of a modified Clostridial toxin disclosed in BoNT/E comprising amino acids 412-426 of SEQID NO: 5; the present specification into the di-chain form. See, e.g., a di-chain loop region of BoNT/F comprising amino acids Steward, L. E. et al., Modified Clostridial Toxins with 429-445 of SEQID NO: 6; a di-chain loop region of BoNT/G Enhanced Targeting Capabilities For Endogenous Clostridial comprising amino acids 436-450 of SEQ ID NO: 7; and a Toxin Receptor Systems, U.S. Patent Publication No. US di-chain loop region of TeNT comprising amino acids 439 2008/0096248 (Apr. 24, 2008); Steward, L. E. et al., Activat 467 of SEQID NO: 8 (Table 2).
TABLE 2 Di-chain Loop Rection of Clost ridial Toxins SEQ Di- chain Loop Region Containing the D Light Chain Naturally-occurring Protease Cleavage Heavy Chain Toxin NO: Region Site Region
BoNT/A 1. NMNFTKLKNFTGLFEFYKLL CWRGIITSKTKSLDKGYNKk - - - -ALNDLC KWNNWDL
BoNT/B 2 KOAYEEISKEHLAVYKIOM CKSWK ------APGIC DWDNEDL
BoNT/C1 3 PALRKWNPENMLYLFTKF CHKAIDGRSLYNK* ------TLDC RELLWKNTDL
BoNT/D 4. PALOKLSSESVVDLFTKV CLRLTKNSR ------DDSTC KWKNNRL
BoNT/E 5. PRIITPITGRGLWKKIIRF CKNIWSWKGIR ------KSIC EINNGEL
BoNT/F 6 PKIIDSIPDKGLWEKIWKF CKSWIPRKGTK ------APPRLC RWNNSEL
BoNT/G 7 KEAYEEISLEHLWIYRIAM CKPWMYKNTGK ------SEQC IWNNEDL
TeNT 8 TNAFRNWDGSGLWSKLIGL CKKIIPPTNIRENLYNRTASLTDLGGELC KIKNEDL
BaNT 9 SRIWGPIPDNGLVERFWGL CKS-IWSKKGTK ------NSLC KWNNRDL
BuNT 2O PRIITPITGRGLWKKIIRF CKN-IWSWKGIR ------KSIC EINNGEL The amino acid sequence displayed are as follows: BoNT/A, residues 410 - 462 of SEQ ID No: 1; BoNTAB, residues 418 - 454 of SEQ ID No. 2; BoNTAC1, residues 419 - 463 of SEQ ID No.: 3; BoNTAD, residues 419 - 458 of SEQ ID No.: 4; BoNTAE, residues 393 - 434 of SEQ ID No: 5; BoNTAF, residues 410 - 453 of SEQ ID No. 6; BoNTAG, residues 419 458 of SEQ ID No: 7; TeNT residues 422 - 475 of SEQ ID No: 8; BaNT residues 4O2 443 of SEQ ID No. 9; and BuNT residues 393 - 434 of SEQ ID No: 1.O. An asterisks (*) indicates the peptide bond that is cleaved by a Clostridial toxin protease. able Clostridial Toxins, U.S. Patent Publication No. US 2008/ 0157. As used herein, the term “endogenous di-chain loop 0032930 (Feb. 7, 2008); Steward, supra, (2007); Dolly, supra, protease cleavage site' is synonymous with a “naturally (2007); Foster, supra, WO 2006/059093 (2006); and Foster, occurring di-chain loop protease cleavage site' and means a naturally occurring protease cleavage site found within the supra, WO 2006/059105 (2006), each of which is hereby di-chain loop region of a naturally occurring Clostridial toxin incorporated by reference in its entirety. and includes, without limitation, naturally occurring 0156. In is envisioned that any and all protease cleavage Clostridial toxin di-chain loop protease cleavage site variants, sites can be used to convert the single-chain polypeptide form Such as, e.g., Clostridial toxin di-chain loop protease cleavage of a Clostridial toxin into the di-chain form, including, with site isoforms and Clostridial toxin di-chain loop protease out limitation, endogenous di-chain loop protease cleavage cleavage site subtypes. Non-limiting examples of an endog sites and exogenous protease cleavage sites. Thus, in an enous protease cleavage site, include, e.g., a BoNT/A di aspect of the invention, a modified Clostridial toxin com chain loop protease cleavage site, a BoNT/B di-chain loop prises, in part, an endogenous protease cleavage site within a protease cleavage site, a BoNT/C1 di-chain loop protease di-chain loop region. In another aspect of the invention, a cleavage site, a BoNT/D di-chain loop protease cleavage site, modified Clostridial toxin comprises, in part, an exogenous a BoNT/E di-chain loop protease cleavage site, a BoNT/F protease cleavage site within a di-chain loop region. As used di-chain loop protease cleavage site, a BoNT/G di-chain loop herein, the term "di-chain loop region' means the amino acid protease cleavage site and a TeNT di-chain loop protease sequence of a Clostridial toxin containing a protease cleavage cleavage site. site used to convert the single-chain form of a Clostridial 0158. As mentioned above, Clostridial toxins are trans toxin into the di-chain form. Non-limiting examples of a lated as a single-chain polypeptide of approximately 150 kDa US 2009/01171.57 A1 May 7, 2009 26 that is Subsequently cleaved by proteolytic Scission within a 0.161 It is also envisioned that an exogenous protease disulfide loop by a naturally-occurring protease. This post cleavage site can be used to convert the single-chain polypep translational processing yields a di-chain molecule compris tide form of a modified Clostridial toxin disclosed in the ing an approximately 50kDa light chain (LC) and an approxi present specification into the di-chain form. As used herein, mately 100 kDa heavy chain (HC) held together by a single the term "exogenous protease cleavage site' is synonymous disulphide bond and noncovalent interactions. While the with a "non-naturally occurring protease cleavage site' or identity of the protease is currently unknown, the di-chain “non-native protease cleavage site' and means a protease loop protease cleavage site for many Clostridial toxins has cleavage site that is not normally present in a di-chain loop been determined. In BoNTs, cleavage at K448-A449 converts region from a naturally occurring Clostridial toxin, with the the single polypeptide form of BoNT/A into the di-chain proviso that the exogenous protease cleavage site is not a form; cleavage at K441-A442 converts the single polypeptide human protease cleavage site or a protease cleavage site that form of BoNT/B into the di-chain form; cleavage at K449 is susceptible to a protease being expressed in the host cell T450 converts the single polypeptide form of BoNT/C1 into that is expressing a construct encoding an activatable the di-chain form; cleavage at R445-D446 converts the single polypeptide disclosed in the present specification. It is envi polypeptide form of BoNT/D into the di-chain form; cleavage Sioned that any and all exogenous protease cleavage sites can at R422-K423 converts the single polypeptide form of be used to convert the single-chain polypeptide form of a BoNT/E into the di-chain form; cleavage at K439-A440 con Clostridial toxin into the di-chain form are useful to practice verts the single polypeptide form of BoNT/F into the di-chain aspects of the present invention. Non-limiting examples of form; and cleavage at K446-S447 converts the single exogenous protease cleavage sites include, e.g., a plant polypeptide form of BoNT/G into the di-chain form. Pro papain cleavage site, an insect papain cleavage site, a crusta teolytic cleavage of the single polypeptide form of TeNT at cian papain cleavage site, an enterokinase cleavage site, a A457-S458 results in the di-chain form. Proteolytic cleavage human rhinovirus 3C protease cleavage site, a human of the single polypeptide form of BaNT at K431-N432 results enterovirus 3C protease cleavage site, a tobacco etch virus in the di-chain form. Proteolytic cleavage of the single (TEV) protease cleavage site, a Tobacco Vein Mottling Virus polypeptide form of BuNT at R422-K423 results in the di (TVMV) cleavage site, a subtilisin cleavage site, a hydroxy chain form. Such a di-chain loop protease cleavage site is lamine cleavage site, or a Caspase 3 cleavage site. operably-linked in-frame to a modified Clostridial toxin as a 0162. It is envisioned that an exogenous protease cleavage fusion protein. However, it should also be noted that addi site of any and all lengths can be useful in aspects of the tional cleavage sites within the di-chain loop also appear to be present invention with the proviso that the exogenous pro cleaved resulting in the generation of a small peptide frag tease cleavage site is capable of being cleaved by its respec ment being lost. As a non-limiting example, BoNT/A single tive protease. Thus, in aspects of this embodiment, an exog chain polypeptide cleave ultimately results in the loss of a ten enous protease cleavage site can have a length of, e.g., at least amino acid fragment within the di-chain loop. 6 amino acids, at least 7 amino acids, at least 8 amino acids, 0159. Thus, in an embodiment, a protease cleavage site at least 9 amino acids, at least 10 amino acids, at least 15 comprising an endogenous Clostridial toxin di-chain loop amino acids, at least 20 amino acids, at least 25 amino acids, protease cleavage site is used to convert the single-chain toxin at least 30 amino acids, at least 40 amino acids, at least 50 into the di-chain form. In aspects of this embodiment, con amino acids, or at least 60 amino acids. In other aspects of this version into the di-chain form by proteolytic cleavage occurs embodiment, an exogenous protease cleavage site can have a from a site comprising, e.g., a BoNT/Adi-chain loop protease length of e.g., at most 6 amino acids, at most 7 amino acids, cleavage site, a BoNTVB di-chain loop protease cleavage site, at most 8 amino acids, at most 9 amino acids, at most 10 a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D amino acids, at most 15 amino acids, at most 20 amino acids, di-chain loop protease cleavage site, a BoNT/E di-chain loop at most 25 amino acids, at most 30 amino acids, at most 40 protease cleavage site, a BoNT/F di-chain loop protease amino acids, at most 50 amino acids, or at most 60 amino cleavage site, a BoNT/G di-chain loop protease cleavage site, acids. a TeNT di-chain loop protease cleavage site, a BaNT di-chain 0163. In an embodiment, an exogenous protease cleavage loop protease cleavage site, or a BuNT di-chain loop protease site is located within the di-chain loop of a modified cleavage site. Clostridial toxin. In aspects of this embodiment, a modified 0160. In other aspects of this embodiment, conversion into Clostridial toxin comprises an exogenous protease cleavage the di-chain form by proteolytic cleavage occurs from a site site comprises, e.g., a plant papain cleavage site, an insect comprising, e.g., a di-chain loop region of BoNT/A compris papain cleavage site, a crustacian papain cleavage site, a ing amino acids 430-454 of SEQID NO: 1; a di-chain loop non-human enterokinase protease cleavage site, a Tobacco region of BoNT/B comprising amino acids 437-446 of SEQ Etch Virus protease cleavage site, a Tobacco Vein Mottling ID NO: 2; a di-chain loop region of BoNT/C1 comprising Virus protease cleavage site, a human rhinovirus 3C protease amino acids 437-453 of SEQID NO:3: a di-chain loop region cleavage site, a human enterovirus 3C protease cleavage site, of BoNT/D comprising amino acids 437-450 of SEQID NO: a Subtilisin cleavage site, a hydroxylamine cleavage site, a 4; a di-chain loop region of BoNT/E comprising amino acids SUMO/ULP-1 protease cleavage site, and a non-human 412-426 of SEQID NO: 5; a di-chain loop region of BoNT/F Caspase 3 cleavage site. In other aspects of this embodiment, comprising amino acids 429-445 of SEQID NO: 6; a di-chain an exogenous protease cleavage site is located within the loop region of BoNT/G comprising amino acids 436-450 of di-chain loop of, e.g., a modified BoNT/A, a modified BoNT/ SEQID NO: 7; or a di-chain loop region of TeNT comprising B, a modified BoNT/C1, a modified BoNT/D, a modified amino acids 439-467 of SEQID NO:8.. a di-chain loop region BoNT/E, a modified BoNT/F, a modified BoNT/G, a modi of BaNT comprising amino acids 421-435 of SEQID NO: 9: fied TeNT, a modified BaNT, or a modified BuNT. or a di-chain loop region of BuNT comprising amino acids 0164. In an aspect of this embodiment, an exogenous pro 412-426 of SEQID NO: 10. tease cleavage site can comprise, e.g., a non-human enteroki US 2009/01171.57 A1 May 7, 2009 27 nase cleavage site is located within the di-chain loop of a located within the di-chain loop of a modified Clostridial modified Clostridial toxin. In other aspects of the embodi toxin comprises the consensus sequence P5-P4-L-F-Q*-G-P ment, an exogenous protease cleavage site can comprise, e.g., (SEQID NO:34), where P4 is G, A, V. L., I, M, S or Tand P5 a bovine enterokinase protease cleavage site located within can any amino acid, with D or E preferred. In other aspects of the di-chain loop of a modified Clostridial toxin. In other the embodiment, an exogenous protease cleavage site can aspects of the embodiment, an exogenous protease cleavage comprise, e.g., a human rhinovirus 3C protease cleavage site site can comprise, e.g., a bovine enterokinase protease cleav located within the di-chain loop of a modified Clostridial age site located within the di-chain loop of a modified toxin comprises SEQ ID NO:35, SEQ ID NO:36, SEQ ID Clostridial toxin comprises SEQ ID NO: 21. In still other NO:37, SEQID NO:38, SEQID NO:39 or SEQID NO:40. aspects of this embodiment, a bovine enterokinase protease In other aspects of the embodiment, an exogenous protease cleavage site is located within the di-chain loop of, e.g., a cleavage site can comprise, e.g., a human rhinovirus 3C pro modified BoNT/A, a modified BoNT/B, a modified BoNT/ tease located within the di-chain loop of a modified C1, a modified BoNT/D, a modified BoNT/E, a modified Clostridial toxin that can be cleaved by PRESCISSIONR). In BoNT/F, a modified BoNT/G, a modified TeNT, a modified still other aspects of this embodiment, a human rhinovirus 3C BaNT, or a modified BuNT. protease cleavage site is located within the di-chain loop of 0.165. In another aspect of this embodiment, an exogenous e.g., a modified BoNT/A, a modified BoNT/B, a modified protease cleavage site can comprise, e.g., a Tobacco Etch BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modi Virus protease cleavage site is located within the di-chain fied BoNT/F, a modified BoNT/G, a modified TeNT, a modi loop of a modified Clostridial toxin. In other aspects of the fied BaNT, or a modified BuNT. embodiment, an exogenous protease cleavage site can com 0.168. In yet another aspect of this embodiment, an exog prise, e.g., a Tobacco Etch Virus protease cleavage site enous protease cleavage site can comprise, e.g., a Subtilisin located within the di-chain loop of a modified Clostridial cleavage site is located within the di-chain loop of a modified toxin comprises the consensus sequence E-P5-P4-Y-P2 Clostridial toxin. In other aspects of the embodiment, an Q*-G (SEQIDNO: 22) or E-P5-P4-Y-P2-Q*-S(SEQID NO: exogenous protease cleavage site can comprise, e.g., a Sub 23), where P2, P4 and P5 can be any amino acid. In other tilisin cleavage site located within the di-chain loop of a aspects of the embodiment, an exogenous protease cleavage modified Clostridial toxin comprises the consensus sequence site can comprise, e.g., a Tobacco Etch Virus protease cleav P6-P5-P4-P3-H*-Y (SEQID NO:41) or P6-P5-P4-P3-Y-H* age site located within the di-chain loop of a modified (SEQ ID NO: 42), where P3, P4 and P5 and P6 can be any Clostridial toxin comprises SEQID NO:24, SEQID NO:25, amino acid. In other aspects of the embodiment, an exog SEQID NO: 26, SEQ ID NO: 27, SEQID NO: 28, SEQ ID enous protease cleavage site can comprise, e.g., a Subtilisin NO:29, SEQID NO:30, SEQID NO:31, SEQID NO:32 or cleavage site located within the di-chain loop of a modified SEQID NO:33. In still other aspects of this embodiment, a Clostridial toxin comprises SEQID NO:43, SEQID NO:44, Tobacco Etch Virus protease cleavage site is located within or SEQ ID NO: 45. In other aspects of the embodiment, an the di-chain loop of, e.g., a modified BoNT/A, a modified exogenous protease cleavage site can comprise, e.g., a Sub BoNT/B, a modified BoNT/C1, a modified BoNT/D, a modi tilisin cleavage site located within the di-chain loop of a fied BoNT/E, a modified BoNT/F, a modified BoNT/G, a modified Clostridial toxin that can be cleaved by GENE modified TeNT, a modified BaNT, or a modified BuNT. NASER). In still other aspects of this embodiment, a subtilisin 0166 In another aspect of this embodiment, an exogenous cleavage site is located within the di-chain loop of, e.g., a protease cleavage site can comprise, e.g., a Tobacco Vein modified BoNT/A, a modified BoNT/B, a modified BoNT/ Mottling Virus protease cleavage site is located within the C1, a modified BoNT/D, a modified BoNT/E, a modified di-chain loop of a modified Clostridial toxin. In other aspects BoNT/F, a modified BoNT/G, a modified TeNT, a modified of the embodiment, an exogenous protease cleavage site can BaNT, or a modified BuNT. comprise, e.g., a Tobacco Vein Mottling Virus protease cleav 0169. In yet another aspect of this embodiment, an exog age site located within the di-chain loop of a modified enous protease cleavage site can comprise, e.g., a hydroxy Clostridial toxin comprises the consensus sequence P6-P5 lamine cleavage site is located within the di-chain loop of a V-R-F-Q*-G (SEQID NO: 113) or P6-P5-V-R-F-Q*-S (SEQ modified Clostridial toxin. In other aspects of the embodi ID NO: 114), where P5 and P6 can be any amino acid. In other ment, an exogenous protease cleavage site can comprise, e.g., aspects of the embodiment, an exogenous protease cleavage a hydroxylamine cleavage site comprising multiples of the site can comprise, e.g., a Tobacco Vein Mottling Virus pro dipeptide N*G. In other aspects of the embodiment, an exog tease cleavage site located within the di-chain loop of a modi enous protease cleavage site can comprise, e.g., a hydroxy fied Clostridial toxin comprises SEQ ID NO: 115, SEQ ID lamine cleavage site located within the di-chain loop of a NO: 116, SEQID NO: 117, or SEQID NO: 118. In still other modified Clostridial toxin comprises SEQID NO:46, or SEQ aspects of this embodiment, a Tobacco Vein Mottling Virus ID NO: 47. In still other aspects of this embodiment, a protease cleavage site is located within the di-chain loop of hydroxylamine cleavage site is located within the di-chain e.g., a modified BoNT/A, a modified BoNT/B, a modified loop of, e.g., a modified BoNT/A, a modified BoNT/B, a BoNT/C1, a modified BoNT/D, a modified BoNT/E, a modi modified BoNT/C1, a modified BoNT/D, a modified BoNT/ fied BoNT/F, a modified BoNT/G, a modified TeNT, a modi E, a modified BoNT/F, a modified BoNT/G, a modified TeNT, fied BaNT, or a modified BuNT. a modified BaNT, or a modified BuNT. 0167. In still another aspect of this embodiment, an exog 0170 In yet another aspect of this embodiment, an exog enous protease cleavage site can comprise, e.g., a human enous protease cleavage site can comprise, e.g., a SUMO/ rhinovirus 3C protease cleavage site is located within the ULP-1 protease cleavage site is located within the di-chain di-chain loop of a modified Clostridial toxin. In other aspects loop of a modified Clostridial toxin. In other aspects of the of the embodiment, an exogenous protease cleavage site can embodiment, an exogenous protease cleavage site can com comprise, e.g., a human rhinovirus 3C protease cleavage site prise, e.g., a SUMO/ULP-1 protease cleavage site located US 2009/01171.57 A1 May 7, 2009 28 within the di-chain loop of a modified Clostridial toxin com a flush cut, exposing the free amino-terminal or carboxyl prising the consensus sequence G-G*-P1-P2'-P3' (SEQ ID terminal of the binding domain necessary for selective bind NO: 112), where P1, P2', and P3' can be any amino acid. In ing of the binding domain to its receptor. other aspects of the embodiment, an exogenous protease 0173 A naturally-occurring protease cleavage site can be cleavage site can comprise, e.g., a SUMO/ULP-1 protease made inoperable by altering at least the two amino acids cleavage site located within the di-chain loop of a modified flanking the peptide bond cleaved by the naturally-occurring Clostridial toxin comprises SEQ ID NO: 48. In still other di-chain loop protease. More extensive alterations can be aspects of this embodiment, a SUMO/ULP-1 protease cleav made, with the proviso that the two cysteine residues of the age site is located within the di-chain loop of, e.g., a modified di-chain loop region remain intact and the region can still BoNT/A, a modified BoNT/B, a modified BoNT/C1, a modi form the disulfide bridge. Non-limiting examples of an amino fied BoNT/D, a modified BoNT/E, a modified BoNT/F, a acid alteration include deletion of an amino acid or replace modified BoNT/G, a modified TeNT, a modified BaNT, or a ment of the original amino acid with a different amino acid. modified BuNT. Thus, in one embodiment, a naturally-occurring protease 0171 In an aspect of this embodiment, an exogenous pro cleavage site is made inoperable by altering the two amino tease cleavage site can comprise, e.g., a non-human Caspase acids flanking the peptide bond cleaved by a naturally-occur 3 cleavage site is located within the di-chain loop of a modi ring protease. In other aspects of this embodiment, a natu fied Clostridial toxin. In other aspects of the embodiment, an rally-occurring protease cleavage site is made inoperable by exogenous protease cleavage site can comprise, e.g., a mouse altering, e.g., at least three amino acids including the two Caspase 3 protease cleavage site located within the di-chain amino acids flanking the peptide bond cleaved by a naturally loop of a modified Clostridial toxin. In other aspects of the occurring protease; at least four amino acids including the embodiment, an exogenous protease cleavage site can com two amino acids flanking the peptide bond cleaved by a natu prise, e.g., a non-human Caspase 3 protease cleavage site rally-occurring protease; at least five amino acids including located within the di-chain loop of a modified Clostridial the two amino acids flanking the peptide bond cleaved by a toxin comprises the consensus sequence D-P3-P2-DP1" naturally-occurring protease; at least six amino acids includ (SEQ ID NO: 119), where P3 can be any amino acid, with E ing the two amino acids flanking the peptide bond cleaved by preferred. P2 can be any amino acid and P1" can any amino a naturally-occurring protease; at least seven amino acids acid, with G or S preferred. In other aspects of the embodi including the two amino acids flanking the peptide bond ment, an exogenous protease cleavage site can comprise, e.g., cleaved by a naturally-occurring protease; at least eight a non-human Caspase 3 protease cleavage site located within amino acids including the two amino acids flanking the pep the di-chain loop of a modified Clostridial toxin comprising tide bond cleaved by a naturally-occurring protease; at least SEQID NO: 120, SEQID NO: 121, SEQID NO: 122, SEQ nine amino acids including the two amino acids flanking the ID NO: 123, SEQID NO: 124, or SEQID NO: 125. In still peptide bond cleaved by a naturally-occurring protease; at other aspects of this embodiment, a bovine enterokinase pro least ten amino acids including the two amino acids flanking tease cleavage site is located within the di-chain loop of, e.g., the peptide bond cleaved by a naturally-occurring protease; at a modified BoNT/A, a modified BoNT/B, a modified BoNT/ least 15 amino acids including the two amino acids flanking C1, a modified BoNT/D, a modified BoNT/E, a modified the peptide bond cleaved by a naturally-occurring protease; or BoNT/F, a modified BoNT/G, a modified TeNT, a modified at least 20 amino acids including the two amino acids flanking BaNT, or a modified BuNT. the peptide bond cleaved by a naturally-occurring protease. 0172 A di-chain loop region is modified to replace a natu 0.174 Instill other aspects of this embodiment, a naturally rally-occurring di-chain loop protease cleavage site for an occurring di-chain protease cleavage site is made inoperable exogenous protease cleavage site. In this modification, the by altering, e.g., at most three amino acids including the two naturally-occurring di-chain loop protease cleavage site is amino acids flanking the peptide bond cleaved by a naturally made inoperable and thus can not be cleaved by its protease. occurring protease; at most four amino acids including the Only the exogenous protease cleavage site can be cleaved by two amino acids flanking the peptide bond cleaved by a natu its corresponding exogenous protease. In this type of modi rally-occurring protease; at most five amino acids including fication, the exogenous protease site is operably-linked in the two amino acids flanking the peptide bond cleaved by a frame to a modified Clostridial toxin as a fusion protein and naturally-occurring protease; at most six amino acids includ the site can be cleaved by its respective exogenous protease. ing the two amino acids flanking the peptide bond cleaved by Replacement of an endogenous di-chain loop protease cleav a naturally-occurring protease; at most seven amino acids age site with an exogenous protease cleavage site can be a including the two amino acids flanking the peptide bond Substitution of the sites where the exogenous site is engi cleaved by a naturally-occurring protease; at most eight neered at the position approximating the cleavage site loca amino acids including the two amino acids flanking the pep tion of the endogenous site. Replacement of an endogenous tide bond cleaved by a naturally-occurring protease; at most di-chain loop protease cleavage site with an exogenous pro nine amino acids including the two amino acids flanking the tease cleavage site can be an addition of an exogenous site peptide bond cleaved by a naturally-occurring protease; at where the exogenous site is engineered at the position differ most ten amino acids including the two amino acids flanking ent from the cleavage site location of the endogenous site, the the peptide bond cleaved by a naturally-occurring protease; at endogenous site being engineered to be inoperable. The loca most 15 amino acids including the two amino acids flanking tion and kind of protease cleavage site may be critical because the peptide bond cleaved by a naturally-occurring protease; or certain binding domains require a free amino-terminal or at most 20 amino acids including the two amino acids flank carboxyl-terminal amino acid. For example, when an opioid ing the peptide bond cleaved by a naturally-occurring pro peptide binding domain is placed between two other domains, tease. e.g., see FIG.4, a criterion for selection of a protease cleavage 0.175. It is understood that a modified Clostridial toxin site could be whether the protease that cleaves its site leaves disclosed in the present specification can optionally further US 2009/01171.57 A1 May 7, 2009 29 comprise a flexible region comprising a flexible spacer. A 0179. It is envisioned that a modified Clostridial toxin flexible region comprising flexible spacers can be used to disclosed in the present specification can comprise a flexible adjust the length of a polypeptide region in order to optimize spacer in any and all locations with the proviso that modified a characteristic, attribute or property of a polypeptide. As a Clostridial toxin is capable of performing the intoxication non-limiting example, a polypeptide region comprising one process. In aspects of this embodiment, a flexible spacer is or more flexible spacers in tandem can be use to better expose positioned between, e.g., an enzymatic domain and a trans a protease cleavage site thereby facilitating cleavage of that location domain, an enzymatic domain and an opioid peptide site by a protease. As another non-limiting example, a binding domain, an enzymatic domain and an exogenous protease cleavage site. In other aspects of this embodiment, a polypeptide region comprising one or more flexible spacers in G-spacer is positioned between, e.g., an enzymatic domain tandem can be use to better present an opioid peptide binding and a translocation domain, an enzymatic domain and an domain, thereby facilitating the binding of that binding opioid peptide binding domain, an enzymatic domain and an domain to its receptor. exogenous protease cleavage site. In other aspects of this 0176 A flexible space comprising a peptide is at least one embodiment, an A-spacer is positioned between, e.g., an amino acid in length and comprises non-charged amino acids enzymatic domain and a translocation domain, an enzymatic with Small side-chain R groups, such as, e.g., glycine, alanine, domain and an opioid peptide binding domain, an enzymatic valine, leucine or serine. Thus, in an embodiment a flexible domain and an exogenous protease cleavage site. spacer can have a length of e.g., at least 1 amino acids, at least 0180. In other aspects of this embodiment, a flexible 2 amino acids, at least 3 amino acids, at least 4 amino acids, spacer is positioned between, e.g., an opioid peptide binding at least 5 amino acids, at least 6 amino acids, at least 7 amino domain and a translocation domain, an opioid peptide binding acids, at least 8 amino acids, at least 9 amino acids, or at least domain and an enzymatic domain, an opioid peptide binding 10 amino acids. In another embodiment, a flexible spacer can domain and an exogenous protease cleavage site. In other have a length of, e.g., at most 1 amino acids, at most 2 amino aspects of this embodiment, a G-spacer is positioned acids, at most 3 amino acids, at most 4 amino acids, at most 5 between, e.g., an opioid peptide binding domain and a trans amino acids, at most 6 amino acids, at most 7 amino acids, at location domain, an opioid peptide binding domain and an most 8 amino acids, at most 9 amino acids, or at most 10 enzymatic domain, an opioid peptide binding domain and an amino acids. In still another embodiment, a flexible spacer exogenous protease cleavage site. In other aspects of this can be, e.g., between 1-3 amino acids, between 2-4 amino embodiment, an A-spacer is positioned between, e.g., an acids, between 3-5 amino acids, between 4-6 amino acids, or opioid peptide binding domain and a translocation domain, an between 5-7 amino acids. Non-limiting examples of a flexible opioid peptide binding domain and an enzymatic domain, an spacer include, e.g., a G-spacers such as GGG, GGGG (SEQ opioid peptide binding domain and an exogenous protease ID NO: 49), and GGGGS (SEQID NO: 50) or an A-spacers cleavage site. such as AAAAAAA (SEQID NO:51) and AAAAV (SEQID 0181. In yet other aspects of this embodiment, a flexible NO: 111). Such a flexible region is operably-linked in-frame spacer is positioned between, e.g., a translocation domain and to the modified Clostridial toxin as a fusion protein. an enzymatic domain, a translocation domain and an opioid 0177 Thus, in an embodiment, a modified Clostridial peptide binding domain, a translocation domain and an exog toxin disclosed in the present specification can further com enous protease cleavage site. In other aspects of this embodi prise a flexible region comprising a flexible spacer. In another ment, a G-spacer is positioned between, e.g., a translocation embodiment, a modified Clostridial toxin disclosed in the domain and an enzymatic domain, a translocation domain and present specification can further comprise flexible region an opioid peptide binding domain, a translocation domain and comprising a plurality of flexible spacers in tandem. In an exogenous protease cleavage site. In other aspects of this aspects of this embodiment, a flexible region can comprise in embodiment, an A-spacer is positioned between, e.g., a trans tandem, e.g., at least 1 G-spacer, at least 2 G-spacers, at least location domain and an enzymatic domain, a translocation 3 G-spacers, at least 4 G-spacers or at least 5 G-spacers. In domain and an opioid peptide binding domain, a translocation other aspects of this embodiment, a flexible region can com domain and an exogenous protease cleavage site. prise in tandem, e.g., at most 1 G-spacer, at most 2G-Spacers, 0182. It is envisioned that a modified Clostridial toxin at most 3 G-spacers, at most 4 G-spacers or at most 5 G-spac disclosed in the present specification can comprise an opioid ers. In still other aspects of this embodiment, a flexible region peptide binding domain in any and all locations with the can comprise in tandem, e.g., at least 1 A-spacer, at least 2 proviso that modified Clostridial toxin is capable of perform A-spacers, at least 3 A-spacers, at least 4A-spacers or at least ing the intoxication process. Non-limiting examples include, 5A-spacers. In still other aspects of this embodiment, a flex locating an opioid peptide binding domain at the amino ter ible region can comprise in tandem, e.g., at most 1 A-spacer, minus of a modified Clostridial toxin; locating an opioid at most 2 A-spacers, at most 3 A-spacers, at most 4A-spacers peptide binding domain between a Clostridial toxin enzy or at most 5 A-spacers. In another aspect of this embodiment, matic domain and a translocation domain of a modified a modified Clostridial toxin can comprise a flexible region Clostridial toxin; and locating an opioid peptide binding comprising one or more copies of the same flexible spacers, domain at the carboxyl terminus of a modified Clostridial one or more copies of different flexible-spacer regions, or any toxin. Other non-limiting examples include, locating an combination thereof. opioid peptide binding domain between a Clostridial toxin 0178. In other aspects of this embodiment, a modified enzymatic domain and a Clostridial toxin translocation Clostridial toxin comprising a flexible spacer can be, e.g., a domain of a modified Clostridial toxin. The enzymatic modified BoNT/A, a modified BoNT/B, a modified BoNT/ domain of naturally-occurring Clostridial toxins contains the C1, a modified BoNT/D, a modified BoNT/E, a modified native start methionine. Thus, in domain organizations where BoNT/F, a modified BoNT/G, a modified TeNT, a modified the enzymatic domain is not in the amino-terminal location an BaNT, or a modified BuNT. amino acid sequence comprising the start methionine should US 2009/01171.57 A1 May 7, 2009 30 be placed in front of the amino-terminal domain. Likewise, 0188 In yet another embodiment, a modified Clostridial where an opioid peptide binding domain is in the amino toxin can comprise an amino to carboxyl single polypeptide terminal position, an amino acid sequence comprising a start linear order comprising a translocation domain, an opioid methionine and a protease cleavage site may be operably peptide binding domain, an exogenous protease cleavage site linked in situations in which an opioid peptide binding and an enzymatic domain (FIG. 4D). In an aspect of this domain requires a free amino terminus, see, e.g., Shengwen embodiment, a modified Clostridial toxin can comprise an Li et al., Degradable Clostridial Toxins, U.S. patent applica amino to carboxyl single polypeptide linear order comprising tion Ser. No. 1 1/572,512 (Jan. 23, 2007), which is hereby a Clostridial toxin translocation domain, an opioid peptide incorporated by reference in its entirety. In addition, it is binding domain, an exogenous protease cleavage site and a known in the art that when adding a polypeptide that is oper Clostridial toxin enzymatic domain. (0189 In still another embodiment, a modified Clostridial ably-linked to the amino terminus of another polypeptide toxin can comprise an amino to carboxyl single polypeptide comprising the start methionine that the original methionine linear order comprising an enzymatic domain, an exogenous residue can be deleted. protease cleavage site, a translocation domain, and an opioid 0183 Thus, in an embodiment, a modified Clostridial peptide binding domain (FIG. 5A). In an aspect of this toxin can comprise an amino to carboxyl single polypeptide embodiment, a modified Clostridial toxin can comprise an linear order comprising an opioid peptide binding domain, a amino to carboxyl single polypeptide linear order comprising translocation domain, an exogenous protease cleavage site a Clostridial toxin enzymatic domain, an exogenous protease and an enzymatic domain (FIG. 3A). In an aspect of this cleavage site, a Clostridial toxin translocation domain, and an embodiment, a modified Clostridial toxin can comprise an opioid peptide binding domain. amino to carboxyl single polypeptide linear order comprising 0190. In still another embodiment, a modified Clostridial an opioid peptide binding domain, a Clostridial toxin trans toxin can comprise an amino to carboxyl single polypeptide location domain, an exogenous protease cleavage site and a linear order comprising a translocation domain, an exogenous Clostridial toxin enzymatic domain. protease cleavage site, an enzymatic domain and an opioid 0184. In another embodiment, a modified Clostridial toxin peptide binding domain, (FIG. 5B). In an aspect of this can comprise an amino to carboxyl single polypeptide linear embodiment, a modified Clostridial toxin can comprise an order comprising an opioid peptide binding domain, an enzy amino to carboxyl single polypeptide linear order comprising matic domain, an exogenous protease cleavage site, and a a Clostridial toxin translocation domain, an opioid peptide translocation domain (FIG. 3B). In an aspect of this embodi binding domain, an exogenous protease cleavage site and a ment, a modified Clostridial toxin can comprise an amino to Clostridial toxin enzymatic domain. carboxyl single polypeptide linear order comprising an 0191) A composition useful in the invention generally is opioid peptide binding domain, a Clostridial toxin enzymatic administered as a pharmaceutical acceptable composition domain, an exogenous protease cleavage site, a Clostridial comprising a modified Clostridial toxin. As used herein, the toxin translocation domain. term “pharmaceutically acceptable' means any molecular 0185. In yet another embodiment, a modified Clostridial entity or composition that does not produce an adverse, aller toxin can comprise an amino to carboxyl single polypeptide gic or other untoward or unwanted reaction when adminis linear order comprising an enzymatic domain, an exogenous tered to an individual. As used herein, the term “pharmaceu protease cleavage site, an opioid peptide binding domain, and tically acceptable composition' is synonymous with a translocation domain (FIG.4A). In an aspect of this embodi “pharmaceutical composition' and means a therapeutically ment, a modified Clostridial toxin can comprise an amino to effective concentration of an active ingredient, such as, e.g., carboxyl single polypeptide linear order comprising a any of the modified Clostridial toxins disclosed in the present Clostridial toxin enzymatic domain, an exogenous protease specification. A pharmaceutical composition comprising a cleavage site, an opioid peptide binding domain, and a modified Clostridial toxin is useful for medical and veterinary Clostridial toxin translocation domain. applications. A pharmaceutical composition may be admin 0186. In yet another embodiment, a modified Clostridial istered to a patientalone, or in combination with other Supple toxin can comprise an amino to carboxyl single polypeptide mentary active ingredients, agents, drugs or hormones. The linear order comprising a translocation domain, an exogenous pharmaceutical compositions may be manufactured using protease cleavage site, an opioid peptide binding domain, and any of a variety of processes, including, without limitation, an enzymatic domain (FIG. 4B). In an aspect of this embodi conventional mixing, dissolving, granulating, dragee-mak ment, a modified Clostridial toxin can comprise an amino to ing, levigating, emulsifying, encapsulating, entrapping, and carboxyl single polypeptide linear order comprising a lyophilizing. The pharmaceutical composition can take any Clostridial toxin translocation domain, an opioid peptide of a variety of forms including, without limitation, a sterile binding domain, an exogenous protease cleavage site and a Solution, Suspension, emulsion, lyophilizate, tablet, pill, pel Clostridial toxin enzymatic domain. let, capsule, powder, syrup, elixir or any other dosage form 0187. In another embodiment, a modified Clostridial toxin suitable for administration. can comprise an amino to carboxyl single polypeptide linear 0.192 Aspects of the present invention provide, in part, a order comprising an enzymatic domain, an opioid peptide composition comprising a modified Clostridial toxin. It is binding domain, an exogenous protease cleavage site, and a envisioned that any of the composition disclosed in the translocation domain (FIG. 4C). In an aspect of this embodi present specification can be useful in a method of treating ment, a modified Clostridial toxin can comprise an amino to urogenital-neurological disorder in a mammal in need carboxyl single polypeptide linear order comprising a thereof, with the proviso that the composition prevents or Clostridial toxin enzymatic domain, an opioid peptide bind reduces a symptom associated with the urogenital-neurologi ing domain, an exogenous protease cleavage site, a cal disorder. Non-limiting examples of compositions com Clostridial toxin translocation domain. prising a modified Clostridial toxin include a modified US 2009/01171.57 A1 May 7, 2009
Clostridial toxin comprising an opioid peptide binding phosphate buffers, neutral buffered saline, phosphate buff domain, a Clostridial toxin translocation domain and a ered saline and borate buffers. It is understood that acids or Clostridial toxin enzymatic domain. It is envisioned that any bases can be used to adjust the pH of a composition as needed. modified Clostridial toxin disclosed in the present specifica Pharmaceutically acceptable antioxidants include, without tion can be used, including those disclosed in, e.g., Steward, limitation, Sodium metabisulfite, Sodium thiosulfate, acetyl supra, (2007); Dolly, supra, (2007); Foster, supra, WO 2006/ cysteine, butylated hydroxyanisole and butylated hydroxy 059093 (2006); Foster, supra, WO 2006/059105 (Jun. 8, toluene. Useful preservatives include, without limitation, 2006). It is also understood that the two or more different benzalkonium chloride, chlorobutanol, thimerosal, phenylm modified Clostridial toxins can be provided as separate com ercuric acetate, phenylmercuric nitrate, a stabilized oxy positions or as part of a single composition. chloro composition, such as, e.g., PURITE(R) and chelants, 0193 It is also envisioned that a pharmaceutical compo such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, and sition comprising a modified Clostridial toxin can optionally CaNaDTPA-bisamide. Tonicity adjustors useful in a pharma include a pharmaceutically acceptable carriers that facilitate ceutical composition include, without limitation, Salts such processing of an active ingredient into pharmaceutically as, e.g., Sodium chloride, potassium chloride, mannitol or acceptable compositions. As used herein, the term “pharma glycerin and other pharmaceutically acceptable tonicity cologically acceptable carrier is synonymous with “pharma adjustor. The pharmaceutical composition may be provided cological carrier and means any carrier that has substantially as a salt and can beformed with many acids, including but not no long term or permanent detrimental effect when adminis limited to, hydrochloric, Sulfuric, acetic, lactic, tartaric, tered and encompasses terms such as “pharmacologically malic. Succinic, etc. Salts tend to be more soluble in aqueous acceptable vehicle, stabilizer, diluent, additive, auxiliary or or other protonic solvents than are the corresponding free excipient. Such a carrier generally is mixed with an active base forms. It is understood that these and other substances compound, or permitted to dilute or enclose the active com known in the art of pharmacology can be included in a phar pound and can be a solid, semi-solid, or liquid agent. It is maceutical composition useful in the invention. understood that the active ingredients can be soluble or can be 0.195. In an embodiment, a composition comprising a delivered as a Suspension in the desired carrier or diluent. Any modified Clostridial toxin is a pharmaceutical composition of a variety of pharmaceutically acceptable carriers can be comprising a modified Clostridial toxin. In aspects of this used including, without limitation, aqueous media Such as, embodiment, a pharmaceutical composition comprising a e.g., water, Saline, glycine, hyaluronic acid and the like; Solid modified Clostridial toxin further comprises a pharmacologi carriers such as, e.g., mannitol, lactose, starch, magnesium cal carrier, a pharmaceutical component, or both a pharma Stearate, sodium saccharin, talcum, cellulose, glucose, cological carrier and a pharmaceutical component. In other Sucrose, magnesium carbonate, and the like; Solvents; disper aspects of this embodiment, a pharmaceutical composition sion media; coatings; antibacterial and antifungal agents; iso comprising a modified Clostridial toxin further comprises at tonic and absorption delaying agents; or any other inactive least one pharmacological carrier, at least one pharmaceutical ingredient. Selection of a pharmacologically acceptable car component, or at least one pharmacological carrier and at rier can depend on the mode of administration. Exceptinsofar least one pharmaceutical component. as any pharmacologically acceptable carrier is incompatible 0.196 Aspects of the present invention provide, in part, an with the active ingredient, its use in pharmaceutically accept urogenital-neurological disorder. As used herein, the term able compositions is contemplated. Non-limiting examples “urogenital-neurological disorder” means an urogenital of specific uses of Such pharmaceutical carriers can be found rooted disorder where at least one of the underlying Symp in PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY toms being treated is due to a nociceptive sensory nerve SYSTEMS (Howard C. Ansel et al., eds., Lippincott Williams & based etiology, Such as, e.g., a spastic dysfunction and/or Wilkins Publishers, 7" ed. 1999); REMINGTON: THE SCIENCE degeneration of the Sacral reflex arcs. Non-limiting examples AND PRACTICE OF PHARMACY (Alfonso R. Gennaro ed., Lip of urogenital-neurological disorders, include, without limita pincott, Williams & Wilkins, 20 ed. 2000); GOODMAN & tion, urinary incontinence, overactive bladder, detrusor dys GILMANS THE PHARMACOLOGICAL BASIS OF THERAPEUTICS function, lower urinary tract dysfunction, urinary retention, (Joel G. Hardman et al., eds., McGraw-Hill Professional, 10" urinary hesitancy, polyuria, nocturia, chronic urinary tract ed. 2001); and HANDBOOK OF PHARMACEUTICAL EXCIPIENTS infection, prostate disorders associated with or without other (Raymond C. Rowe et al., APhA Publications, 4" edition urogenital disorders, uterine disorders associated with or 2003). These protocols are routine procedures and any modi without other urogenital disorders, and urogenital disorders fications are well within the scope of one skilled in the art and associated with neurogenic dysfunction (Such as, e.g., uro from the teaching herein. genital disorders associated with Parkinson's Disease, mul 0194 It is further envisioned that a pharmaceutical com tiple Sclerosis, spina bifida, transverse myelitis, stroke, spinal position disclosed in the present specification can optionally cord injury, spasm reflex, and a neurologic lesion of the spinal include, without limitation, other pharmaceutically accept cord or brain), and other Such urogenital disorders of a noci able components (orpharmaceutical components), including, ceptive sensory nerve-based etiology. without limitation, buffers, preservatives, tonicity adjusters, 0.197 An individual's ability to hold urine and maintain salts, antioxidants, osmolality adjusting agents, physiological continence depends on normal function of the lower urinary Substances, pharmacological Substances, bulking agents, tract, the kidneys, and the nervous system. The individual emulsifying agents, wetting agents, Sweetening or flavoring must also have a physical and psychological ability to recog agents, and the like. Various buffers and means for adjusting nize and appropriately respond to the urge to urinate. The pH can be used to prepare a pharmaceutical composition bladder's ability to fill and store urine requires a functional disclosed in the present specification, provided that the result sphincter muscle (which controls the flow of urine out of the ing preparation is pharmaceutically acceptable. Such buffers body) and a stable bladder wall muscle (detrusor). Normal include, without limitation, acetate buffers, citrate buffers, bladder function is dependent on the nerves that sense the US 2009/01171.57 A1 May 7, 2009 32 fullness of the bladder and on those that trigger the muscle cause cannot be identified. Although urge incontinence may movements that either empty it or retain urine. The process of occur in anyone at any age, it is more common in women and urination involves two phases: 1) filling and storage of blad the elderly. Urge incontinence is also known as irritable blad der and 2) emptying of bladder. During the filling and storage der, spasmodic bladder, and unstable bladder. phase, the bladder stretches So it can hold the increasing 0201 Overflow urinary incontinence happens when small amount of urine. The bladder of an average person can hold amounts of urine leak from a bladder that is always full. In 350 mL to 550 mL of urine. Generally, the reflex to urinate is older men, this can occur when the flow of urine from the triggered when the bladder of an individual when approxi bladder is blocked, usually by an enlarged prostate. It can mately 200 mL of urine collects in the bladder. The emptying Sometimes be prevented by medication when early symptoms phase requires that the detrusor muscle contract, forcing urine of prostate enlargement, such as frequent urination, appear. out of the bladder through the urethra. The sphincter muscle Some people with diabetes also have overflow incontinence. must relax at the same time, so that urine can flow out of the Mixed urinary incontinence describes a disorder where an body. The bladder, internal sphincters, and external sphinc individual exhibits symptoms associated with both stress ters may all be affected by nociceptive sensory nerve-based incontinence and urge incontinence. Continuous urinary disorders that create abnormalities in bladder function. The incontinence is the complaint of continuous leakage. damage can cause the bladder to be underactive, in which it is 0202 Thus in embodiment, a mammal suffering from uri unable to contract and unable to empty completely, or it can nary incontinence is treated with a composition comprising a be overactive, in which it contracts too quickly or frequently. therapeutically effective amount of a modified Clostridial 0198 One type of urogenital-neurological disorder is uri toxin where Such administration reduces a symptom associ nary incontinence. Urinary incontinence is the inability to ated with the urinary incontinence. In an aspect of this control the passage of urine. This can range from an occa embodiment, a mammal Suffering from stress incontinence is sional leakage of urine, to a complete inability to hold any treated with a composition comprising a therapeutically urine. Urinary incontinence can becaused by abnormalities in effective amount of a modified Clostridial toxin where such bladder capacity or malfunction of control mechanisms such administration reduces a symptom associated with the stress as the bladder neck and/or external urethral sphincter muscle incontinence. In another aspect of this embodiment, a mam that are important for the bladder's storage function. The mal Suffering from urge incontinence is treated with a com many types of urinary incontinence. position comprising a therapeutically effective amount of a 0199 Stress incontinence is a type of urinary incontinence modified Clostridial toxin where such administration reduces in which the strength of the muscles (urethral sphincter) that a symptom associated with the urge incontinence. In still help control urination is reduced as a result of weakened another aspect of this embodiment, a mammal Suffering from pelvic muscles that support the bladder and urethra or overflow urinary incontinence is treated with a composition because of malfunction of the urethral sphincter. The weak comprising a therapeutically effective amount of a modified ness may be caused by prior injury to the urethral area, neu Clostridial toxin where such administration reduces a symp rological injury, some medications, or after Surgery of the tom associated with the overflow urinary incontinence. In a prostate or pelvic area. The sphincter is not able to prevent further aspect of this embodiment, a mammal Suffering from urine flow when there is increased pressure from the abdomen mixed urinary incontinence is treated with a composition Such as during certain activities like coughing, Sneezing, comprising a therapeutically effective amount of a modified laughing, or exercise. Stress urinary incontinence is the most Clostridial toxin where such administration reduces a symp common type of urinary incontinence in women. Studies tom associated with the mixed urinary incontinence. In a have shown about 50% of all women have occasional urinary further aspect of this embodiment, a mammal Suffering from incontinence, and as many as 10% have frequent inconti continuous urinary incontinence is treated with a composition nence. Nearly 20% of women over age 75 experience daily comprising a therapeutically effective amount of a modified urinary incontinence. Stress incontinence is often seen in Clostridial toxin where such administration reduces a symp women who have had multiple pregnancies and vaginal child tom associated with the continuous urinary incontinence. births, whose bladder, urethra, or rectal wall stick out into the 0203. Another type of urogenital-neurological disorder is vaginal space (pelvic prolapse). overactive bladder. Overactive bladder is increased urinary 0200 Urge incontinence is a type of urinary incontinence urgency, with or without urge urinary incontinence, usually that involves a strong, Sudden need to urinate, followed by with frequency and nocturia. The individual may report instant bladder contraction and involuntary loss of urine symptoms of urinary urgency (the Sudden, intense desire to which results in leakage. There is not enough time between urinate immediately), urinary frequency (the need to urinate when an individual Suffering from urge incontinence recog more times than is normal), enuresis (any involuntary loss of nizes the need to urinate and when urination actually occurs. urine), polyuria, nocturia, and/or urinary incontinence. Thus, Urge incontinence is leakage of urine due to bladder muscles overactive bladder describes a bladder that contracts more that contract inappropriately. Often these contractions occur often than it should, so that a person feels the need to urinate regardless of the amount of urine that is in the bladder. Urge more frequently and/or urgently than necessary and is char incontinence may result from neurological injuries (such as acterized by uncontrolled, frequent expulsion of urine from spinal cord injury or stroke), neurological dysfunction (Such the bladder. An overactive bladder usually, but not always, as, e.g., Parkinson's Disease and multiple Sclerosis), infec causes urinary incontinence. Individuals with overactive tion, bladder cancer, bladderstones, bladder inflammation, or bladder may go to the bathroom very often, e.g., every two bladder outlet obstruction. In men, urge incontinence may be hours during the day and night, and may even wet the bed. due to neurological disease or bladder changes caused by Often, a strong urge to Void is experienced when only a small benign prostatic hypertrophy (BPH) or bladder outlet amount of urine is in the bladder. There may be reduced obstruction from an enlarged prostate. The majority of cases bladder capacity and incomplete emptying of urine. An over of urge incontinence are idiopathic, which means a specific active bladder can be caused by interruptions in the nerve US 2009/01171.57 A1 May 7, 2009
pathways to the bladder occurring above the sacrum. For embodiment, a mammal Suffering from overactive bladder is example, spastic bladder may be caused by an inability of the treated with a composition comprising a therapeutically detrusor muscle of the bladder to inhibit emptying contrac effective amount of a modified Clostridial toxin where such tions until a reasonable amount of urine has accumulated. As administration reduces urinary incontinence. such, overactive bladder is often associated with detrusor 0206. Another type of urogenital-neurological disorder is overactivity, a pattern of bladder muscle contraction observed detrusor dysfunction, including, without limitation, detrusor during urodynamics. Overactive bladder can also be caused overactivity, detrusor instability, and detrusor-sphincter dys by urinary tract infection, outflow obstruction and stress synergia. One kind of detrusor dysfunction is detrusor over incontinence. Sometimes no cause is found, and Such idio activity or involuntary detrusor contractions (previously pathic cases may be due to anxiety or aging. Symptoms termed detrusor hyperreflexia). Detrusor overactivity include the need to urinate may times throughout the day and involves increased involuntary contractions of the detrusor night, the sensation of having to urinate immediately, and/or muscle during the filling phase which may be spontaneous or the sudden leakage of urine from the bladder. provoked resulting in uninhibitable bladder contractions. The 0204 Diseases extrinsic to the bladder may also cause the muscle contraction patterns of detrusor overactivity include, symptoms of overactive bladder. In the male patient, the without limitation, phasic detrusor overactivity and terminal extrinsic disorder most often responsible for overactive blad detrusor overactivity. Detrusor overactivity can be either idio der is bladder outlet obstruction (BOO). Disorders extrinsic pathic in nature or they can be caused by non-neurogenic or to the bladder in the female patient include urethral diverticu neurogenic conditions. Symptoms of detrusor overactivity lum, retroverted uterus, pelvic prolapse (including cystocele), include, without limitation, uninhibitable bladder contrac gravid uterus, and loss or reduction of estrogen. Disorders tions, urinary urgency, urinary frequency, enuresis, polyuria, extrinsic to the bladder common to both men and woman nocturia, and/or urinary incontinence. Another kind of detru include pelvic mass, physiologic nocturnal diuresis, and sor dysfunction is detrusor instability. Detrusor instability polyuria caused by factors such as excessive fluid intake, involves uncontrolled involuntary contractions of the detru diuretic use, or diabetes. Neuromuscular disorders may also Sor muscle resulting in uninhibitable bladder contractions account for the overactive bladder. Neurogenic disorders irrespective of bladder capacity. Symptoms of detrusor insta resulting from nerve damage to sensory nerves can also cause bility include, without limitation, uninhibitable bladder con overactive bladder, including, without limitation, Parkinson tractions, urinary urgency, urinary frequency, enuresis, poly disease, multiple Sclerosis, spina bifida, cervical Stenosis, uria, nocturia, and/or urinary incontinence. Another kind of spinal cord injury, diabetic neuropathy, pelvic Surgery, or detrusor dysfunction is detrusor-sphincter dyssynergia invertebral disc herniation, hydrocephalus, stroke, spinal (DSD). Detrusor-sphincter dyssynergia occurs when the con cord injuries and lesions of the spinal cord or brain. Bladder traction of the detrusor musculature is not coordinated with aging may also account for these symptoms. A patient history the relaxation of the sphincter thereby preventing the urethra of pelvic trauma, pelvic radiation, or bladder, prostate, or from relaxing completely during voiding. Symptoms of urethral Surgery should also be considered when seeking to detrusor-sphincter dyssynergia include, without limitation, determine the etiology of the overactive bladder. urine flow interruption, raised detrusor pressure and/or uri 0205 Thus in embodiment, a mammal suffering from nary retention. DSD can be caused as a consequence of a overactive bladder is treated with a composition comprising a neurological condition Such as spinal injury or multiple scle therapeutically effective amount of a modified Clostridial OS1S. toxin where Such administration reduces a symptom associ 0207 Thus in embodiment, a mammal suffering from ated with the overactive bladder. In an aspect of this embodi detrusor dysfunction is treated with a composition compris ment, a mammal Suffering from overactive bladder is treated ing a therapeutically effective amount of a modified with a composition comprising a therapeutically effective Clostridial toxin where such administration reduces a symp amount of a modified Clostridial toxin where such adminis tom associated with the detrusor dysfunction. In an aspect of tration reduces incontinence. In an aspect of this embodi this embodiment, a mammal Suffering from detrusor dysfunc ment, a mammal Suffering from overactive bladder is treated tion is treated with a composition comprising a therapeuti with a composition comprising a therapeutically effective cally effective amount of a modified Clostridial toxin where amount of a modified Clostridial toxin where such adminis such administration reduces uninhibitable bladder contrac tration reduces urinary frequency. In another aspect of this tions. In another aspect of this embodiment, a mammal Suf embodiment, a mammal Suffering from overactive bladder is fering from detrusor dysfunction is treated with a composi treated with a composition comprising a therapeutically tion comprising a therapeutically effective amount of a effective amount of a modified Clostridial toxin where such modified Clostridial toxin where such administration reduces administration reduces urinary urgency. In another aspect of urinary frequency. In another aspect of this embodiment, a this embodiment, a mammal Suffering from overactive blad mammal Suffering from detrusor dysfunction is treated with a der is treated with a composition comprisingatherapeutically composition comprising atherapeutically effective amount of effective amount of a modified Clostridial toxin where such a modified Clostridial toxin where such administration administration reduces enuresis. In another aspect of this reduces urinary urgency. In yet another aspect of this embodi embodiment, a mammal Suffering from overactive bladder is ment, a mammal Suffering from detrusor dysfunction is treated with a composition comprising a therapeutically treated with a composition comprising a therapeutically effective amount of a modified Clostridial toxin where such effective amount of a modified Clostridial toxin where such administration reduces polyuria. In yet another aspect of this administration reduces enuresis. In yet another aspect of this embodiment, a mammal Suffering from overactive bladder is embodiment, a mammal Suffering from detrusor dysfunction treated with a composition comprising a therapeutically is treated with a composition comprising a therapeutically effective amount of a modified Clostridial toxin where such effective amount of a modified Clostridial toxin where such administration reduces nocturia. In yet another aspect of this administration reduces polyuria. In yet another aspect of this US 2009/01171.57 A1 May 7, 2009 34 embodiment, a mammal Suffering from detrusor dysfunction another aspect of this embodiment, a mammal Suffering from is treated with a composition comprising a therapeutically detrusor instability is treated with a composition comprising effective amount of a modified Clostridial toxin where such a therapeutically effective amount of a modified Clostridial administration reduces nocturia. In yet another aspect of this toxin where Such administration reduces urinary frequency. embodiment, a mammal Suffering from detrusor dysfunction In another aspect of this embodiment, a mammal Suffering is treated with a composition comprising a therapeutically from detrusor instability is treated with a composition com effective amount of a modified Clostridial toxin where such prising a therapeutically effective amount of a modified administration reduces urinary incontinence. In still another Clostridial toxin where such administration reduces urinary aspect of this embodiment, a mammal Suffering from detrusor urgency. In yet another aspect of this embodiment, a mammal dysfunction is treated with a composition comprising athera suffering from detrusor instability is treated with a composi peutically effective amount of a modified Clostridial toxin tion comprising a therapeutically effective amount of a modi where such administration reduces urine flow interruption. In fied Clostridial toxin where such administration reduces still another aspect of this embodiment, a mammal Suffering from detrusor dysfunction is treated with a composition com enuresis. In yet another aspect of this embodiment, a mammal prising a therapeutically effective amount of a modified suffering from detrusor instability is treated with a composi Clostridial toxin where such administration reduces detrusor tion comprising a therapeutically effective amount of a modi pressure. In still another aspect of this embodiment, a mam fied Clostridial toxin where such administration reduces mal suffering from detrusor dysfunction is treated with a polyuria. In yet another aspect of this embodiment, a mammal composition comprising atherapeutically effective amount of suffering from detrusor instability is treated with a composi a modified Clostridial toxin where such administration tion comprising a therapeutically effective amount of a modi reduces urinary retention. fied Clostridial toxin where such administration reduces noc 0208. In another embodiment, a mammal suffering from turia. In yet another aspect of this embodiment, a mammal detrusor overactivity is treated with a composition compris suffering from detrusor instability is treated with a composi ing a therapeutically effective amount of a modified tion comprising a therapeutically effective amount of a modi Clostridial toxin where such administration reduces a symp fied Clostridial toxin where such administration reduces uri tom associated with the detrusor overactivity. In an aspect of nary incontinence. this embodiment, a mammal Suffering from detrusor overac 0210. In still another embodiment, a mammal suffering tivity is treated with a composition comprising a therapeuti from detrusor-sphincter dyssynergia is treated with a compo cally effective amount of a modified Clostridial toxin where sition comprising a therapeutically effective amount of a such administration reduces uninhibitable bladder contrac modified Clostridial toxin where such administration reduces tions. In another aspect of this embodiment, a mammal Suf a symptom associated with the detrusor-sphincter dyssyner fering from detrusor overactivity is treated with a composi gia. In an aspect of this embodiment, a mammal Suffering tion comprising a therapeutically effective amount of a from detrusor-sphincter dyssynergia is treated with a compo modified Clostridial toxin where such administration reduces sition comprising a therapeutically effective amount of a urinary frequency. In another aspect of this embodiment, a modified Clostridial toxin where such administration reduces mammal suffering from detrusor overactivity is treated with a urine flow interruption. In another aspect of this embodiment, composition comprising atherapeutically effective amount of a mammal Suffering from detrusor-sphincter dyssynergia is a modified Clostridial toxin where such administration treated with a composition comprising a therapeutically reduces urinary urgency. In yet another aspect of this embodi effective amount of a modified Clostridial toxin where such ment, a mammal Suffering from detrusor overactivity is administration reduces detrusor pressure. In yet another treated with a composition comprising a therapeutically aspect of this embodiment, a mammal Suffering from detru effective amount of a modified Clostridial toxin where such sor-sphincter dyssynergia is treated with a composition com administration reduces enuresis. In yet another aspect of this prising a therapeutically effective amount of a modified embodiment, a mammal Suffering from detrusor overactivity Clostridial toxin where such administration reduces urinary is treated with a composition comprising a therapeutically retention. effective amount of a modified Clostridial toxin where such 0211 Another type of urogenital-neurological disorder is administration reduces polyuria. In yet another aspect of this a lower urinary tract dysfunction (LUTD). See e.g., Paul embodiment, a mammal Suffering from detrusor overactivity Abrams et al., The Standardisation of Terminology of Lower is treated with a composition comprising a therapeutically Urinary Tract Function. Report from the Standardisation effective amount of a modified Clostridial toxin where such Subcommittee of the International Continence Society, 21 administration reduces nocturia. In yet another aspect of this Neurourol Urodyn. 167-178 (2002), which is hereby incor embodiment, a mammal Suffering from detrusor overactivity porated by reference in its entirety. Lower urinary tract dys is treated with a composition comprising a therapeutically functions manifest three general types of symptoms: storage, effective amount of a modified Clostridial toxin where such Voiding, and post-micturition symptoms. Storage symptoms administration reduces urinary incontinence. are experienced during the storage phase of the bladder and 0209. In yet another embodiment, a mammal suffering include, without limitation, urinary urgency, urinary fre from detrusor instability is treated with a composition com quency, enuresis, polyuria, nocturia increased bladder sensa prising a therapeutically effective amount of a modified tion, decreased bladder sensation, absent bladder sensation, Clostridial toxin where such administration reduces a symp non-specific bladder sensation, and/or urinary incontinence. tom associated with the detrusor instability. In an aspect of Voiding symptoms are experienced during the Voiding phase. this embodiment, a mammal Suffering from detrusor instabil Symptoms include, without limitation, reduced urine flow, ity is treated with a composition comprising a therapeutically splitting or spraying of urine, intermittent urine flow, urinary effective amount of a modified Clostridial toxin where such hesitancy, strained effort to void urine, and/or terminal administration reduces uninhibitable bladder contractions. In dribble of urine. Post-micturition symptoms are experienced US 2009/01171.57 A1 May 7, 2009
immediately after micturition and include, without limita flow and/or infection. Either chronic or acute retention may tion, sensation of incomplete emptying and/or post-micturi lead to incontinence due to leakage of urine from an overfull tion dribble. bladder. 0212. Thus in embodiment, a mammal suffering from a 0214 Thus in embodiment, a mammal suffering from uri lower urinary tract dysfunction is treated with a composition nary retention is treated with a composition comprising a comprising a therapeutically effective amount of a modified therapeutically effective amount of a modified Clostridial Clostridial toxin where such administration reduces a symp toxin where Such administration reduces a symptom associ tom associated with the lower urinary tract dysfunction. In an ated with the urinary retention. In an aspect of this embodi aspect of this embodiment, a mammal Suffering from a lower ment, a mammal Suffering from urinary retention is treated urinary tract dysfunction is treated with a composition com with a composition comprising a therapeutically effective prising a therapeutically effective amount of a modified amount of a modified Clostridial toxin where such adminis Clostridial toxin where Such administration reduces storage tration reduces urinary urgency. In another aspect of this symptoms. In aspects of this embodiment, the storage symp embodiment, a mammal Suffering from urinary retention is tom reduced is urinary urgency, urinary frequency, enuresis, treated with a composition comprising a therapeutically polyuria, nocturia increased bladder sensation, decreased effective amount of a modified Clostridial toxin where such bladder sensation, absent bladder sensation, non-specific administration reduces urinary frequency. In yet another bladder sensation, or urinary incontinence. In another aspect aspect of this embodiment, a mammal Suffering from urinary of this embodiment, a mammal Suffering from a lower urinary retention is treated with a composition comprising a thera tract dysfunction is treated with a composition comprising a peutically effective amount of a modified Clostridial toxin therapeutically effective amount of a modified Clostridial where such administration increases bladder capacity. In still toxin where such administration reduces voiding symptoms. another aspect of this embodiment, a mammal Suffering from In aspects of this embodiment, the Voiding symptom reduced urinary retention is treated with a composition comprising a is reduced urine flow, splitting or spraying of urine, intermit therapeutically effective amount of a modified Clostridial tenturine flow, urinary hesitancy, strained effort to void urine, toxin where Such administration reduces urinary inconti or terminal dribble of urine. In yet another aspect of this nence. In still another aspect of this embodiment, a mammal embodiment, a mammal Suffering from a lower urinary tract Suffering from urinary retention is treated with a composition dysfunction is treated with a composition comprising athera comprising a therapeutically effective amount of a modified peutically effective amount of a modified Clostridial toxin Clostridial toxin where such administration restores normal where such administration reduces post-micturition symp urine flow. toms. In aspects of this embodiment, the post-micturition 0215. In another embodiment, a mammal suffering from symptom reduced is sensation of incomplete emptying or acute urinary retention is treated with a composition compris post-micturition dribble. ing a therapeutically effective amount of a modified 0213 Another type of urogenital-neurological disorder is Clostridial toxin where such administration reduces a symp urinary retention. Urinary retention is the inability to pass tomassociated with the acute urinary retention. In yet another urine from the bladder and may be either an acute or chronic embodiment, a mammal Suffering from chronic urinary reten condition. Normally, the reflex to urinate is triggered when tion is treated with a composition comprising a therapeuti the bladder fills to approximately 300-500 mL. The bladder is cally effective amount of a modified Clostridial toxin where then emptied when the contraction of the bladder wall forces Such administration reduces a symptom associated with the urine out through the urethra. The bladder, internal sphinc chronic urinary retention. ters, and external sphincters may all be affected by disorders 0216. Another type of urogenital-neurological disorder is that create abnormalities in bladder function resulting in uri urinary hesitancy. Urinary hesitancy is difficulty starting or nary retention. Urinary retention can result either from loss of maintaining a urinary stream. This problem affects people of bladder muscle contracting performance or loss of appropri all ages and occurs in both sexes, but it is most common in ate coordination between the bladder muscle and the urethral older men with enlarged prostate glands. Urinary hesitancy sphincter muscle. The inability to properly relax the urinary usually comes on gradually. It sometimes goes unnoticed sphincter muscles causing difficulty in emptying the bladder, until urinary retention (complete inability to urinate) pro which can lead to urinary retention. Often, a strong urge to duces distention and discomfort in the bladder. Almost all Void is experienced when only a small amount of urine is in older men have some degree of difficulty in starting urination, the bladder. In addition, there may be reduced bladder capac dribbling, or decreased force of the urinary stream. Urinary ity and incomplete emptying of urine. Urinary retention may hesitancy can be caused by benign prostatic hyperplasia (en also be caused by difficulty in relaxing the urinary sphincter larged prostate), urinary tract infection, especially if chronic muscle because the sphincter may be spastic. Alternatively, and recurrent, prostatitis (inflammation or infection of the the bladder neck may be hypertrophied. Other causes of uri prostate gland), drugs (some cold remedies, some nasal nary retention include interruptions in the nerve pathways to decongestants, tricyclic antidepressants, and anticholinergics the bladder occurring above the sacrum. This nerve damage which may be used for incontinence), shy or bashful bladder results in a loss of sensation and motor control and is often syndrome in younger people (unable to urinate when another seen in stroke, Parkinson's disease, spina bifida, diabetes, person is in the room), and neurological disorders. pelvic Surgery, or invertebral discherniation, and most forms 0217 Thus in embodiment, a mammal suffering from uri of spinal cord injuries. Sometimes no cause is found, and Such nary hesitancy is treated with a composition comprising a idiopathic cases may be due to anxiety or aging. Urinary therapeutically effective amount of a modified Clostridial retention can also occur by a blockage to the flow of urine due toxin where Such administration reduces a symptom associ to prostate enlargement or urinary tract stones. Another type ated with the urinary hesitancy. In an aspect of this embodi of urinary retention disorder is stones, which block the uri ment, a mammal Suffering from urinary hesitancy is treated nary tract of an individual thereby causing stoppage of urine with a composition comprising a therapeutically effective US 2009/01171.57 A1 May 7, 2009 36 amount of a modified Clostridial toxin where such adminis empty fully due to Such conditions as benign prostatic hyper tration reduces urinary urgency. In another aspect of this plasia, prostatitis, and urethral strictures. Other irritating embodiment, a mammal Suffering from urinary hesitancy is symptoms may include painful urination (dysuria), which treated with a composition comprising a therapeutically may be a result of a urinary tract infection (UTI) caused by effective amount of a modified Clostridial toxin where such urine being held too long in the bladder. UTI with fever is a administration reduces urinary frequency. In yet another sign of potential severe kidney infection (pyelonephritis) and aspect of this embodiment, a mammal Suffering from urinary is a more worrisome situation as it may result in permanent hesitancy is treated with a composition comprising a thera damage of the kidney(s). Another type of urinary tract infec peutically effective amount of a modified Clostridial toxin tion is vesicoureteral reflux (VUR). Vesicoureteral reflux is an where such administration increases bladder capacity. In still abnormal backup of urine from the bladder to the kidney(s) another aspect of this embodiment, a mammal Suffering from that occurs as a means of releasing high pressure within the urinary hesitancy is treated with a composition comprising a bladder. AUTI is of particular concern as VUR may place the therapeutically effective amount of a modified Clostridial patient at significant risk for a severe kidney infection by toxin where Such administration reduces urinary inconti transporting infected bladder urine directly to the kidney(s). nence. In still another aspect of this embodiment, a mammal 0221) Thus in embodiment, a mammal suffering from Suffering from urinary hesitancy is treated with a composition chronic urinary tract infection is treated with a composition comprising a therapeutically effective amount of a modified comprising a therapeutically effective amount of a modified Clostridial toxin where such administration restores normal Clostridial toxin where such administration reduces a symp urine flow. tom associated with the chronic urinary tract infection. In an 0218. Another type of urogenital-neurological disorder is aspect of this embodiment, a mammal Suffering from dysuria polyuria. Polyuria is when a person releases abnormally is treated with a composition comprising a therapeutically excessive Volume of urine each day. An excessive Volume of effective amount of a modified Clostridial toxin where such urination for an adult would be at least 2.5 liters of urine per administration reduces a symptom associated with the dys day. Polyuria is a fairly common symptom, which is often uria. In an aspect of this embodiment, a mammal Suffering noticed when you have to get up to use the bathroom at night. from vesicoureteral reflux is treated with a composition com Thus in embodiment, a mammal Suffering from polyuria is prising a therapeutically effective amount of a modified treated with a composition comprising a therapeutically Clostridial toxin where such administration reduces a symp effective amount of a modified Clostridial toxin where such tom associated with the vesicoureteral reflux. administration reduces a symptom associated with the poly 0222. Other types of urogenital-neurological disorders are U18 disorders associated with prostate disorders. The prostate is a 0219. Another type of urogenital-neurological disorder is partially glandular and partially fibromuscular organ of the nocturia. Nocturia is excessive urination at night, Such as by male reproductive system that that produces the fluid that waking up several times during the night to urinate. Normally, carries sperm during ejaculation. It surrounds the urethra, the urine decreases in amount and become more concentrated at tube through which urine passes out of the body. One type of night. That means, most people can sleep 6 to 8 hours without prostate disorder is benign prostatic hyperplasia (BPH). Dur having to urinate. But, persons with nocturiaget up more than ingaging, the prostate tends to enlarge (hypertrophy) and this once during the night to urinate. Because of this, those who enlarged prostate is often called benign prostatic hyperplasia have excessive urination at night often have disrupted sleep (BPH) or benign prostatic hypertrophy. Prostatic enlargement cycles. Causes include benign prostatic hyperplasia, certain can lead to urethral obstruction and Voiding dysfunction drugs including diuretics, cardiac glycosides, demeclocy because the enlarged gland can press on the urethra. BPH is cline, lithium, methoxyflurane, phenytoin, propoxyphene, not cancer, and it does not raise your risk for prostate cancer. and excessive vitamin D. chronic or recurrent urinary tract One type of prostate disorder is prostatitis. Prostatitis is an infection, chronic renal failure, congestive heart failure, cys inflammation of the prostate gland. Prostatitis include acute titis, diabetes, drinking too much fluid before bedtime, par and chronic bacterial prostatitis and inflammation not caused ticularly coffee, caffeinated beverages, or alcohol, and by bacterial infection (abacterial prostatitis). One type of obstructive sleep apnea and other sleeping disorders. Thus in prostate disorder is prostatodynia. Prostatodynia is a type of embodiment, a mammal Suffering from nocturia is treated inflammation of the prostate not due to bacterial infection that with a composition comprising a therapeutically effective may be caused by abnormal nerves or muscles in the region. amount of a modified Clostridial toxin where such adminis Prostatodynia is typically a chronic, painful disease. The tration reduces a symptom associated with the nocturia. symptoms (including chills, fever, pain in the lower back and 0220 Another type of urogenital-neurological disorder is genital area, body aches, burning or painful urination, and the chronic urinary tract infection (recurrent infection). Chronic frequent and urgent need to urinate) characteristically go urinary tract infection (UTI) is a bacterial infection of the away and then come back without warning. bladder or lower urinary tract (urethra) that lasts for a long 0223 Thus in embodiment, a mammal suffering from a time. Most urinary tract infections occur in the lower urinary urogenital-neurological disorder associated with a prostate tract, which includes the bladder and urethra. The condition disorder is treated with a composition comprising atherapeu occurs when the normally clean lower urinary tract is infected tically effective amount of a modified Clostridial toxin where by bacteria and becomes inflamed. Urinary tract infections Such administration reduces a symptom associated with the are very common. Most of the time, symptoms of a urinary urogenital-neurological disorder associated with the prostate tract infection disappear within 24–48 hours after treatment disorder. In another aspect of this embodiment, a mammal begins. However, if the condition occurs more than twice in 6 Suffering from urogenital-neurological disorder associated months, lasts longer than 2 weeks, or does not respond to with benign prostatic hyperplasia is treated with a composi usual treatment, it is considered chronic. The elderly are at tion comprising a therapeutically effective amount of a modi increased risk for such infections because the bladder doesn't fied Clostridial toxin where such administration reduces a US 2009/01171.57 A1 May 7, 2009 37 symptom associated with the urogenital-neurological disor lem(s) such as endometriosis (abnormalities in the lining of der associated with benign prostatic hyperplasia. In yet the uterus), adenomyosis (nonmalignant growth of the another aspect of this embodiment, a mammal Suffering from endometrium into the muscular layer of the uterus), pelvic urogenital-neurological disorder associated with prostatitis is inflammatory disease, uterine fibroids, cervical narrowing, treated with a composition comprising a therapeutically uterine malposition, pelvic tumors or an IUD (intra-uterine effective amount of a modified Clostridial toxin where such device). This condition usually occurs in older women. administration reduces a symptom associated with the uro 0226. Thus in embodiment, a mammal suffering from a genital-neurological disorder associated with prostatitis. In urogenital-neurological disorder associated with a uterine still another aspect of this embodiment, a mammal Suffering disorder is treated with a composition comprising atherapeu from urogenital-neurological disorder associated with pros tically effective amount of a modified Clostridial toxin where tatodynia is treated with a composition comprising a thera Such administration reduces a symptom associated with the peutically effective amount of a modified Clostridial toxin urogenital-neurological disorder associated with the uterine where Such administration reduces a symptom associated disorder. In an aspect of this embodiment, a mammal Suffer with the urogenital-neurological disorder associated with ing from endometriosis is treated with a composition com prostatodynia. prising a therapeutically effective amount of a modified 0224. In another embodiment, a mammal suffering from a Clostridial toxin where such administration reduces a symp prostate disorder is treated with a composition comprising a tom associated with the endometriosis. In an aspect of this therapeutically effective amount of a modified Clostridial embodiment, a mammal Suffering from dysmenorrhea is toxin where Such administration reduces a symptom associ treated with a composition comprising a therapeutically ated with the prostate disorder. In an aspect of this embodi effective amount of a modified Clostridial toxin where such ment, a mammal Suffering from benign prostatic hyperplasia administration reduces a symptom associated with the dys is treated with a composition comprising a therapeutically menorrhea. effective amount of a modified Clostridial toxin where such 0227 Other types of urogenital-neurological disorders are administration reduces a symptom associated with the benign urogenital-neurological disorders associated with neurogenic prostatic hyperplasia. In yet another aspect of this embodi dysfunction. Thus in embodiment, a mammal Suffering from ment, a mammal Suffering from prostatitis is treated with a a urogenital-neurological disorder associated with a neuro composition comprising atherapeutically effective amount of genic dysfunction is treated with a composition comprising a a modified Clostridial toxin where such administration therapeutically effective amount of a modified Clostridial reduces a symptom associated with the prostatitis. In still toxin where such administration reduces a symptom associ another aspect of this embodiment, a mammal Suffering from ated with the urogenital-neurological disorder associated prostatodynia is treated with a composition comprising a with the neurogenic dysfunction. In an aspect of this embodi therapeutically effective amount of a modified Clostridial ment, a mammal Suffering from a urogenital-neurological toxin where Such administration reduces a symptom associ disorder associated with Parkinson's Disease is treated with a ated with the prostatodynia. composition comprising atherapeutically effective amount of 0225. Other types of urogenital-neurological disorders are a modified Clostridial toxin where such administration disorders associated with uterine disorders. The uterus is a reduces a symptom associated with the urogenital-neurologi hollow, muscular pear-shaped female reproductive organ in cal disorder associated with Parkinson's Disease. In another which the fertilized Zygote implants and develops into the aspect of this embodiment, a mammal Suffering from a uro fetus. The uterus comprises a corpus made up of two layers of genital-neurological disorder associated with multiple scle tissue, fundus, isthmus, and cervix located between the uri rosis is treated with a composition comprising a therapeuti nary bladder and the rectum in the pelvic cavity of female cally effective amount of a modified Clostridial toxin where mammals. One type of uterine disorder is endometriosis. Such administration reduces a symptom associated with the Endometriosis is a condition in which the tissue that lines the urogenital-neurological disorder associated with multiple inside of the uterus (called the endometrium or endometrial Sclerosis. In yet another aspect of this embodiment, a mam lining) is found growing in other areas outside of the uterus mal Suffering from a urogenital-neurological disorder asso (commonly the ovaries, fallopian tubes, outer Surface of the ciated with spina bifida is treated with a composition com uterus, outer Surface of the intestines, and nearby structures of prising a therapeutically effective amount of a modified the pelvis). This condition often causes severe pain within the Clostridial toxin where such administration reduces a symp lower abdomen and pelvis that may be associated with your tom associated with the urogenital-neurological disorder periods each month. The symptoms of endometriosis include associated with spina bifida. In yet another aspect of this pain before and during menstrual periods, pain at the time of embodiment, a mammal Suffering from a urogenital-neuro ovulation, pain during or after sexual activity, heavy or irregu logical disorder associated with transverse myelitis is treated lar bleeding, fatigue, pain with bowel movements at the time with a composition comprising a therapeutically effective of the period, pain with urination. Another type of uterine amount of a modified Clostridial toxin where such adminis disorder is dysmenorrhea. Dysmenorrhea is the pain or dis tration reduces a symptom associated with the urogenital comfort (menstrual cramps) during or just before a menstrual neurological disorder associated with transverse myelitis. In period. There are two types of dysmenorrheal, primary dys yet another aspect of this embodiment, a mammal Suffering menorrhea and secondary dysmenorrhea. Primary dysmen from a urogenital-neurological disorder associated with orrhea is severe, disabling cramps without underlying illness. stroke is treated with a composition comprising a therapeuti Symptoms may include backache, leg pain, nausea, vomiting, cally effective amount of a modified Clostridial toxin where diarrhea, headache, and dizziness. This kind of dysmenorrhea Such administration reduces a symptom associated with the usually affects young woman within two years of the onset of urogenital-neurological disorder associated with stroke. In menstruation and lasts one or two days each month. Second still another aspect of this embodiment, a mammal Suffering ary dysmenorrhea is cramps caused by another medical prob from a urogenital-neurological disorder associated with a US 2009/01171.57 A1 May 7, 2009
spinal cord injury is treated with a composition comprising a ods known to those of skill in the art, including, but not therapeutically effective amount of a modified Clostridial restricted to, encapsulation in liposomes, by ionophoresis, or toxin where Such administration reduces a symptom associ by incorporation into other vehicles, such as hydrogels, ated with the urogenital-neurological disorder associated cyclodextrins, biodegradable nanocapsules, and bioadhesive with the spinal cord injury. In still another aspect of this microspheres, or by proteinaceous vectors. Delivery mecha embodiment, a mammal Suffering from a urogenital-neuro nisms for administering a composition comprising a modified logical disorder associated with a spasm refleX is treated with Clostridial toxin to a patient are described in, e.g., Leonid a composition comprising a therapeutically effective amount Beigelman et al., Compositions for the Delivery of Nega of a modified Clostridial toxin where such administration tively Charged Molecules, U.S. Pat. No. 6,395,713 (May 28, reduces a symptom associated with the urogenital-neurologi 2002); and Achim Aigner, Delivery Systems for the Direct cal disorder associated with the spasm reflex. In a further Application of siRNAs to Induce RNA Interference (RNAi) in aspect of this embodiment, a mammal Suffering from a uro vivo, 2006(716559) J. Biomed. Biotech. 1-15 (2006); Con genital-neurological disorder associated with a neurologic trolled Drug Delivery. Designing Technologies for the Future lesion of the spinal cord or brain is treated with a composition (Kinam Park & Randy J. Mrsny eds. American Chemical comprising a therapeutically effective amount of a modified Association, 2000); Vernon G. Wong & Mae W. L. Hu, Meth Clostridial toxin where such administration reduces a symp ods for Treating Inflammation-mediated Conditions of the tom associated with the urogenital-neurological disorder Eye, U.S. Pat. No. 6,726,918 (Apr. 27, 2004); David A. Weber associated with the neurologic lesion of the spinal cord or et al., Methods and Apparatus for Delivery of Ocular brain. Implants, U.S. Patent Publication No. US2004/0054374 0228 Aspects of the present invention provide, in part, a (Mar. 18, 2004); Thierry Nivaggioli et al., Biodegradable mammal. A mammal includes a human, and a human can be Ocular Implant, U.S. Patent Publication No. US2004/ a patient. Other aspects of the present invention provide, in 0.137059 (Jul. 15, 2004); Patrick M. Hughes et al., Anti part, an individual. An individual includes a human, and a Angiogenic Sustained Release Intraocular Implants and human can be a patient. Related Methods, U.S. patent application Ser. No. 1 1/364, 0229. Aspects of the present invention provide, in part, 687 (Feb. 27, 2006); and Patrick M. Hughes et al., Sustained administering a composition comprising a modified Release Intraocular Drug Delivery Systems, U.S. Patent Pub Clostridial toxin. As used herein, the term “administering lication 2006/0182783 (Aug. 17, 2006), each of which is means any delivery mechanism that provides a composition hereby incorporated by reference in its entirety. comprising a modified Clostridial toxin to a patient that 0232 A composition comprising a modified Clostridial potentially results in a clinically, therapeutically, or experi toxin as disclosed in the present specification can also be mentally beneficial result. A modified Clostridial toxin can be administered to a patient using a gene therapy approach by delivered to a patient using a cellular uptake approach where expressing a modified Clostridial toxin within in a cell mani a modified Clostridial toxin is delivered intracellular or a gene festing a nerve-based etiology that contributes to aurogenital therapy approach where a modified Clostridial toxin is neurological disorder. A modified Clostridial toxin can be express derived from precursor RNAs expressed from an expressed from nucleic acid molecules operably-linked to an expression vectors. expression vector, see, e.g., P. D. Good et al., Expression of 0230. A composition comprising a modified Clostridial Small, Therapeutic RNAs in Human Cell Nuclei, 4(1) Gene toxin as disclosed in the present specification can be admin Ther. 45-54 (1997); James D. Thompson, Polymerase III istered to a mammal using a cellular uptake approach. Admin based expression of therapeutic RNAs, U.S. Pat. No. 6,852, istration of a composition comprising a modified Clostridial 535 (Feb. 8, 2005); Macie Wiznerowicz et al., Tuning toxin using a cellular uptake approach comprise a variety of Silence: Conditional Systems for RNA Interference, 3(9) Nat. enteral or parenteral approaches including, without limita Methods 682-688m (2006); Ola Snave and John J. Rossi, tion, oral administration in any acceptable form, such as, e.g., Expressing Short Hairpin RNAi in vivo, 3(9) Nat. Methods tablet, liquid, capsule, powder, or the like; topical adminis 689-698 (2006); and Charles X. Li et al., Delivery of RNA tration in any acceptable form, Such as, e.g., drops, spray, Interference, 5(18) Cell Cycle 2103-2109 (2006). A person of creams, gels or ointments; intravascular administration in any ordinary skill in the art would realize that any modified acceptable form, such as, e.g., intravenous bolus injection, Clostridial toxin can be expressed in eukaryotic cells using an intravenous infusion, intra-arterial bolus injection, intra-arte appropriate expression vector. rial infusion and catheter instillation into the vasculature; per 0233 Expression vectors capable of expressing a modi and intra-tissue administration in any acceptable form, Such fied Clostridial toxin can provide persistent or stable expres as, e.g., intraperitoneal injection, intramuscular injection, sion of the modified Clostridial toxin in a cell manifesting a Subcutaneous injection, Subcutaneous infusion, intraocular nerve-based etiology that contributes to a urogenital-neuro injection, retinal injection, or Sub-retinal injection or epidural logical disorder. Alternatively, expression vectors capable of injection; intravesicular administration in any acceptable expressing a modified Clostridial toxin can provide for tran form, Such as, e.g., catheter instillation; and by placement sient expression of the modified Clostridial toxin in a cell device. Such as, e.g., an implant, a patch, a pellet, a catheter, manifesting a nerve-based etiology that contributes to a uro an osmotic pump, a Suppository, a bioerodible delivery sys genital-neurological disorder. Such transiently expressing tem, a non-bioerodible delivery system or another implanted vectors can be repeatedly administered as necessary. A modi extended or slow release system. An exemplary list of biode fied Clostridial toxin-expressing vectors can be administered gradable polymers and methods of use are described in, e.g., by a delivery mechanism and route of administration dis Handbook of Biodegradable Polymers (Abraham J. Domb et cussed above, by administration to target cells ex-planted al., eds. Overseas Publishers Association, 1997). from a patient followed by reintroduction into the patient, or 0231. A composition comprising a modified Clostridial by any other means that would allow for introduction into the toxin can be administered to a mammal by a variety of meth desired target cell, see, e.g., Larry A. Couture and Dan T. US 2009/01171.57 A1 May 7, 2009 39
Stinchcomb, Anti-gene Therapy. The Use of Ribozymes to 0237. In an embodiment, a composition comprising a Inhibit Gene Function, 12(12) Trends Genet. 510-515 (1996). modified Clostridial toxin is administered systemically to a 0234. The actual delivery mechanism used to administer a mammal. In another embodiment, a composition comprising composition comprising a modified Clostridial toxin to a a modified Clostridial toxin is administered locally to a mam mammal can be determined by a person of ordinary skill in mal. In an aspect of this embodiment, a composition com the art by taking into account factors, including, without prising a modified Clostridial toxin is administered to the limitation, the type of urogenital-neurological disorder, the bladder of a mammal. In another aspect of this embodiment, location of the urogenital-neurological disorder, the cause of a composition comprising a modified Clostridial toxin is the urogenital-neurological disorder, the severity of the uro administered to the prostate of a mammal. In another aspect genital-neurological disorder, the degree of relief desired, the of this embodiment, a composition comprising a modified duration of relief desired, the particular modified Clostridial Clostridial toxin is administered to the uterus of a mammal. toxin used, the rate of excretion of the modified Clostridial 0238 Aspects of the present invention provide, in part, toxin used, the pharmacodynamics of the modified administering a therapeutically effective amount of a compo sition comprising a modified Clostridial toxin. As used Clostridial toxin used, the nature of the other compounds to herein, the term “therapeutically effective amount' is synony be included in the composition, the particular route of admin mous with “therapeutically effective dose' and when used in istration, the particular characteristics, history and risk fac reference to treating an urogenital-neurological disorder tors of the patient, such as, e.g., age, weight, general health means the minimum dose of a modified Clostridial toxin and the like, or any combination thereof. necessary to achieve the desired therapeutic effect and 0235. In an embodiment, a composition comprising a includes a dose Sufficient to reduce a symptom associated modified Clostridial toxin is administered to the site to be with an urogenital-neurological disorder. In aspects of this treated by injection. In aspects of this embodiment, injection embodiment, a therapeutically effective amount of a compo of a composition comprising a modified Clostridial toxin is sition comprising a modified Clostridial toxin reduces a by, e.g., intramuscular injection, Subdermal injection, or der symptom associated with an urogenital-neurological disorder mal injection. In aspects of this embodiment, injection of a by, e.g., at least 30%, at least 40%, at least 50%, at least 60%, composition comprising a modified Clostridial toxin is into at least 70%, at least 80%, at least 90% or at least 100%. In the lower urinary tract, including the bladder wall, the urinary other aspects of this embodiment, a therapeutically effective sphincter or bladder neck. amount of a composition comprising a modified Clostridial 0236 A composition comprising a modified Clostridial toxin reduces a symptom associated with an urogenital-neu toxin can be administered to a mammal using a variety of rological disorderby, e.g., at most 30%, at most 40%, at most routes. Routes of administration suitable for a method of 50%, at most 60%, at most 70%, at most 80%, at most 90% or treating an urogenital-neurological disorder as disclosed in at most 100%. In yet other aspects of this embodiment, a the present specification include both local and systemic therapeutically effective amount of a composition comprising administration. Local administration results in significantly a modified Clostridial toxin reduces a symptom associated more delivery of a composition to a specific location as com with an urogenital-neurological disorder by, e.g., about 10% pared to the entire body of the mammal, whereas, systemic to about 100%, about 10% to about 90%, about 10% to about administration results in delivery of a composition to essen 80%, about 10% to about 70%, about 10% to about 60%, tially the entire body of the patient. Routes of administration about 10% to about 50%, about 10% to about 40%, about 20% Suitable for a method of treating an urogenital-neurological to about 100%, about 20% to about 90%, about 20% to about disorder as disclosed in the present specification also include 80%, about 20% to about 20%, about 20% to about 60%, both central and peripheral administration. Central adminis about 20% to about 50%, about 20% to about 40%, about 30% tration results in delivery of a composition to essentially the to about 100%, about 30% to about 90%, about 30% to about central nervous system of the patient and includes, e.g., 80%, about 30% to about 70%, about 30% to about 60%, or intrathecal administration, epidural administration as well as about 30% to about 50%. As used herein, the term “about a cranial injection or implant. Peripheral administration when qualifying a value of a stated item, number, percentage, results in delivery of a composition to essentially any area of or term refers to a range of plus or minus ten percent of the a patient outside of the central nervous system and encom value of the stated item, percentage, parameter, or term. In passes any route of administration other than direct adminis still other aspects of this embodiment, a therapeutically effec tration to the spine or brain. The actual route of administration tive amount of the modified Clostridial toxin is the dosage of a composition comprising a modified Clostridial toxin Sufficient to inhibit neuronal activity for, e.g., at least one used in a mammal can be determined by a person of ordinary week, at least one month, at least two months, at least three skill in the art by taking into account factors, including, with months, at least four months, at least five months, at least six out limitation, the type of urogenital-neurological disorder, months, at least seven months, at least eight months, at least the location of the urogenital-neurological disorder, the cause nine months, at least ten months, at least eleven months, or at of the urogenital-neurological disorder, the severity of the least twelve months. urogenital-neurological disorder, the degree of relief desired, 0239. The actual therapeutically effective amount of a the duration of relief desired, the particular modified composition comprising a modified Clostridial toxin to be Clostridial toxin used, the rate of excretion of the modified administered to a mammal can be determined by a person of Clostridial toxin used, the pharmacodynamics of the modi ordinary skill in the art by taking into account factors, includ fied Clostridial toxin used, the nature of the other compounds ing, without limitation, the type of urogenital-neurological to be included in the composition, the particular route of disorder, the location of the urogenital-neurological disorder, administration, the particular characteristics, history and risk the cause of the urogenital-neurological disorder, the severity factors of the mammal. Such as, e.g., age, weight, general of the urogenital-neurological disorder, the degree of relief health and the like, or any combination thereof. desired, the duration of relief desired, the particular modified US 2009/01171.57 A1 May 7, 2009 40
Clostridial toxin used, the rate of excretion of the modified composition comprising a modified Clostridial toxin can be, Clostridial toxin used, the pharmacodynamics of the modi e.g., at least 0.00001 mg/kg, at least 0.0001 mg/kg, at least fied Clostridial toxin used, the nature of the other compounds 0.001 mg/kg, at least 0.01 mg/kg, at least 0.1 mg/kg, or at least to be included in the composition, the particular route of 1 mg/kg. In yet other aspects of this embodiment, a therapeu administration, the particular characteristics, history and risk tically effective amount of a composition comprising a modi factors of the patient, such as, e.g., age, weight, general health fied Clostridial toxin can be, e.g., at most 0.00001 mg/kg, at and the like, or any combination thereof. Additionally, where most 0.0001 mg/kg, at most 0.001 mg/kg, at most 0.01 mg/kg, repeated administration of a composition comprising a modi at most 0.1 mg/kg, or at most 1 mg/kg. fied Clostridial toxin is used, the actual effect amount of a 0242 Dosing can be single dosage or cumulative (serial composition comprising a modified Clostridial toxin will fur dosing), and can be readily determined by one skilled in the ther depend upon factors, including, without limitation, the art. For instance, treatment of an urogenital-neurological dis frequency of administration, the half-life of the composition order may comprise a one-time administration of an effective comprising a modified Clostridial toxin, or any combination dose of a composition comprising a modified Clostridial thereof. In is known by a person of ordinary skill in the art that toxin. As a non-limiting example, an effective dose of a com an effective amount of a composition comprising a modified position comprising a modified Clostridial toxin can be Clostridial toxin can be extrapolated from in vitro assays and administered once to a patient, e.g., as a single injection or in vivo administration studies using animal models prior to deposition at or near the site exhibiting a symptom of an administration to humans. Wide variations in the necessary urogenital-neurological disorder. Alternatively, treatment of effective amount are to be expected in view of the differing an urogenital-neurological disorder may comprise multiple efficiencies of the various routes of administration. For administrations of an effective dose of a composition com instance, oral administration generally would be expected to prising a modified Clostridial toxin carried out over a range of require higher dosage levels than administration by intrave time periods, such as, e.g., daily, once every few days, weekly, nous or intravitrealinjection. Variations in these dosage levels monthly or yearly. As a non-limiting example, a composition can be adjusted using standard empirical routines of optimi comprising a modified Clostridial toxin can be administered Zation, which are well-known to a person of ordinary skill in once or twice yearly to a mammal. The timing of administra the art. The precise therapeutically effective dosage levels and tion can vary from mammal to mammal, depending upon patterns are preferably determined by the attending physician Such factors as the severity of a mammal’s symptoms. For in consideration of the above-identified factors. example, an effective dose of a composition comprising a 0240. As a non-limiting example, when administering a modified Clostridial toxin can be administered to a mammal composition comprising a modified Clostridial toxin to a once a month for an indefinite period of time, or until the mammal, a therapeutically effective amount generally is in patient no longer requires therapy. A person of ordinary skill the range of about 1 fig to about 3.0 mg. In aspects of this in the art will recognize that the condition of the mammal can embodiment, an effective amount of a composition compris be monitored throughout the course of treatment and that the ing a modified Clostridial toxin can be, e.g., about 100 fg to effective amount of a composition comprising a modified about 3.0 mg, about 100 pg to about 3.0 mg, about 100 ng to Clostridial toxin that is administered can be adjusted accord about 3.0 mg. or about 100 lug to about 3.0 mg. In other ingly. aspects of this embodiment, an effective amount of a compo 0243 A composition comprising a modified Clostridial sition comprising a modified Clostridial toxin can be, e.g., toxin as disclosed in the present specification can also be about 100 fg to about 750 ug, about 100 pg to about 750 ug, administered to a mammal in combination with other thera about 100 ng to about 750 ug, or about 1 lug to about 750 ug. peutic compounds to increase the overall therapeutic effect of In yet other aspects of this embodiment, a therapeutically the treatment. The use of multiple compounds to treat an effective amount of a composition comprising a modified indication can increase the beneficial effects while reducing Clostridial toxin can be, e.g., at least 1 fg, at least 250 fg, at the presence of side effects. least 500 fg, at least 750 fg, at least 1 pg, at least 250 pg, at 0244 Aspects of the present invention can also be least 500 pg, at least 750 pg, at least 1 ng, at least 250 ng, at described as follows: least 500 ng, at least 750 ng, at least 1 Jug, at least 250 ug, at 0245 1. A method of treating urogenital-neurological dis least 500 ug, at least 750 ug, or at least 1 mg. In still other order in a mammal, the method comprising the step of aspects of this embodiment, a therapeutically effective administering to the mammal in need thereofatherapeuti amount of a composition comprising a modified Clostridial cally effective amount of a composition including a modi toxin can be, e.g., at most 1 fg, at most 250 fg, at most 500 fg, fied Clostridial toxin comprising an opioid peptide binding at most 750 fg, at most 1 pg, at most 250 pg. at most 500 pg. domain, a Clostridial toxin translocation domain and a at most 750 pg, at most 1 ng, at most 250 ng, at most 500 ng, Clostridial toxin enzymatic domain, wherein administra at most 750 ng, at most lug, at least 250 ug, at most 500 ug, tion of the composition reduces a symptom of the urogeni at most 750 ug, or at most 1 mg. tal-neurological disorder, thereby treating the mammal. 0241 As another non-limiting example, when administer 0246 2. The method of 1, wherein the modified Clostridial ing a composition comprising a modified Clostridial toxin to toxin comprises a linear amino-to-carboxyl single a mammal, a therapeutically effective amount generally is in polypeptide order of 1) the Clostridial toxin enzymatic the range of about 0.00001 mg/kg to about 3.0 mg/kg. In domain, the Clostridial toxin translocation domain, the aspects of this embodiment, an effective amount of a compo opioid peptide binding domain, 2) the Clostridial toxin sition comprising a modified Clostridial toxin can be, e.g., enzymatic domain, the opioid peptide binding domain, the about 0.0001 mg/kg to about 0.001 mg/kg, about 0.03 mg/kg Clostridial toxin translocation domain, 3) the opioid pep to about 3.0 mg/kg, about 0.1 mg/kg to about 3.0 mg/kg, or tide binding domain, the Clostridial toxin translocation about 0.3 mg/kg to about 3.0 mg/kg. In yet other aspects of domain, and the Clostridial toxin enzymatic domain, 4) the this embodiment, a therapeutically effective amount of a opioid peptide binding domain, the Clostridial toxin enzy US 2009/01171.57 A1 May 7, 2009 41
matic domain, the Clostridial toxin translocation domain, BoNT/B translocation domain, a BoNT/C1 translocation 5) the Clostridial toxin translocation domain, the domain, a BoNTVD translocation domain, a BoNT/E trans Clostridial toxin enzymatic domain and the opioid peptide location domain, a BoNT/F translocation domain, a binding domain, or 6) the Clostridial toxin translocation BoNT/G translocation domain, a TeNT translocation domain, the opioid peptide binding domain and the domain, a BaNT translocation domain, or a BuNT trans Clostridial toxin enzymatic domain. location domain. 0247 3. The method of 1, wherein the opioid peptide 0261) 17. The method of 1, wherein the Clostridial toxin binding domain is an enkephalin, a BAM22 peptide, an enzymatic domain is a BoNT/A enzymatic domain, a endomorphin, an endorphin, a dynorphin, a nociceptin or a BoNT/B enzymatic domain, a BoNT/C1 enzymatic hemorphin. domain, a BoNT/D enzymatic domain, a BoNT/E enzy 0248 4. The method of 3, wherein the enkephalin is a matic domain, a BoNT/F enzymatic domain, a BoNT/G Leu-enkephalin, a Met-enkephalin, a Met-enkephalin enzymatic domain, a TeNT enzymatic domain, a BaNT MRGL or a Met-enkephalin MRF. enzymatic domain, or a BuNT enzymatic domain. 0249 5. The method of 3, wherein the enkephalin com 0262. 18. The method of 1, wherein the urogenital-neuro prises SEQID NO:52, SEQID NO:53, SEQID NO:54 or logical disorder is urinary incontinence, overactive blad SEQ ID NO:55. der, detrusor dysfunction, lower urinary tract dysfunction, (0250) 6. The method of 3, wherein the BAM22 peptide is urinary retention, urinary hesitancy, polyuria, nocturia, a BAM22 peptide (1-12), a BAM22 peptide (6-22), a chronic urinary tract infection, an urogenital disorder asso BAM22 peptide (8-22) or a BAM22 peptide (1-22) ciated with a prostate disorder, an urogenital disorder asso 0251 7. The method of 3, wherein the BAM22 peptide ciated with a uterine disorder, or an urogenital disorder comprises amino acids 1-12, amino acids 6-22, amino associated with a neurogenic dysfunction. acids 8-22 or amino acids 1-22 of SEQID NO: 56; amino 0263. 19. The method of 18, wherein the urinary inconti acids 1-12, amino acids 6-22, amino acids 8-22 or amino nence is an urge urinary incontinence, a stress urinary acids 1-22 of SEQ ID NO: 57; amino acids 1-12, amino incontinence, an overflow urinary incontinence, a mixed acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ urinary incontinence, or a continuous urinary inconti ID NO: 58; amino acids 1-12, amino acids 6-22, amino CCC. acids 8-22 or amino acids 1-22 of SEQID NO. 59; amino 0264. 20. The method of 18, wherein the detrusor dysfunc acids 1-12, amino acids 6-22, amino acids 8-22 or amino tion is a detrusor overactivity, a detrusor instability, or a acids 1-22 of SEQID NO: 60 or amino acids 1-12, amino detrusor-sphincter dyssynergia. acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ 0265 21. The method of 18, wherein the urogenital disor ID NO: 61 der associated with a prostate disorder is an urogenital 0252 8. The method of 3, wherein the endomorphin is an disorder associated with benign prostatic hyperplasia, an endomorphin-1 or an endomorphin-2. urogenital disorder associated with prostatitis, or an uro 0253) 9. The method of 3, wherein the endomorphin com genital disorder associated with prostatodynia. prises SEQID NO: 62 or SEQID NO: 63. 0266 22. The method of 18, wherein the urogenital disor 0254 10. The method of 3, wherein the endorphin an der associated with a neurogenic dysfunction is an urogeni endorphin-C, a neoendorphin-C., an endorphin-fi, a neoen tal disorder associated with Parkinson's Disease, an uro dorphin-for an endorphin-Y. genital disorder associated with multiple Sclerosis, an 0255 11. The method of 3, wherein the endorphin com urogenital disorder associated with spina bifida, an uro prises SEQID NO: 64, SEQID NO: 65, SEQID NO: 66, genital disorder associated with transverse myelitis, an SEQ ID NO: 67, SEQID NO: 68 or SEQID NO: 69. urogenital disorder associated with stroke, an urogenital 0256 12. The method of 3, wherein the dynorphin is a disorder associated with a spinal cord injury, an urogenital dynorphin A, a dynorphin B (leumorphin) or a rimorphin. disorder associated with a spasm reflex, an urogenital dis 0257 13. The method of 3, wherein the dynorphin com order associated with a neurologic lesion of the spinal cord, prises SEQID NO: 70, SEQID NO: 71, SEQID NO: 72, or an urogenital disorder associated with a neurologic SEQID NO: 73, SEQID NO: 74, SEQID NO: 75, SEQID lesion of the brain. NO:76, SEQID NO: 77, SEQID NO: 78, SEQID NO: 79, 0267. 23. A method of treating urogenital-neurological SEQID NO:80, SEQID NO:81, SEQID NO: 82, SEQID disorder in a mammal, the method comprising the step of NO: 83, SEQID NO:84, SEQID NO: 85, SEQID NO:86, administering to the mammal in need thereofatherapeuti SEQID NO:87, SEQID NO: 88, SEQID NO: 89, SEQID cally effective amount of a composition including a modi NO:90, SEQID NO:91, SEQID NO:92, SEQID NO:93, fied Clostridial toxin comprising an opioid peptide binding SEQID NO:94, SEQID NO:95, SEQID NO:96, SEQID domain, a Clostridial toxin translocation domain, a NO:97, SEQID NO: 98, SEQID NO: 99 or SEQID NO: Clostridial toxin enzymatic domain, and an exogenous pro 1OO. tease cleavage site, wherein administration of the compo 0258 14. The method of 3, wherein the nociceptin is a sition reduces a symptom of the urogenital-neurological nociceptin RK, a nociceptin, a neuropeptide 1, a neuropep disorder, thereby treating the mammal. tide 2 or a neuropeptide 3. 0268 24. The method of 23, wherein the modified 0259 15. The method of 3, wherein the nociceptin com Clostridial toxin comprises a linear amino-to-carboxyl prises SEQ ID NO: 101, SEQID NO: 102, SEQ ID NO: single polypeptide order of 1) the Clostridial toxin enzy 103, SEQID NO: 104, SEQID NO: 105, SEQIDNO: 106, matic domain, the exogenous protease cleavage site, the SEQ ID NO: 107, SEQID NO: 108, SEQID NO: 109 or Clostridial toxin translocation domain, the opioid peptide SEQ ID NO: 110. binding domain, 2) the Clostridial toxin enzymatic 0260 16. The method of 1, wherein the Clostridial toxin domain, the exogenous protease cleavage site, the opioid translocation domain is a BoNT/A translocation domain, a peptide binding domain, the Clostridial toxin translocation US 2009/01171.57 A1 May 7, 2009 42
domain, 3) the opioid peptide binding domain, the 0281 37. The method of 25, wherein the nociceptin com Clostridial toxin translocation domain, the exogenous pro prises SEQ ID NO: 101, SEQID NO: 102, SEQ ID NO: tease cleavage site and the Clostridial toxin enzymatic 103, SEQID NO: 104, SEQIDNO: 105, SEQIDNO: 106, domain, 4) the opioid peptide binding domain, the SEQ ID NO: 107, SEQ ID NO: 108, SEQID NO: 109 or Clostridial toxin enzymatic domain, the exogenous pro SEQID NO: 110. tease cleavage site, the Clostridial toxin translocation 0282. 38. The method of 23, wherein the Clostridial toxin domain, 5) the Clostridial toxin translocation domain, the translocation domain is a BoNT/A translocation domain, a exogenous protease cleavage site, the Clostridial toxin BoNT/B translocation domain, a BoNT/C1 translocation enzymatic domain and the opioid peptide binding domain, domain, a BoNTVD translocation domain, a BoNT/E trans or 6) the Clostridial toxin translocation domain, the exog location domain, a BoNT/F translocation domain, a enous protease cleavage site, the opioid peptide binding BoNT/G translocation domain, a TeNT translocation domain and the Clostridial toxin enzymatic domain. domain, a BaNT translocation domain, or a BuNT trans 0269. 25. The method of 23, wherein the opioid peptide location domain. binding domain is an enkephalin, a BAM22 peptide, an 0283 39. The method of 23, wherein the Clostridial toxin endomorphin, an endorphin, a dynorphin, a nociceptin or a enzymatic domain is a BoNT/A enzymatic domain, a hemorphin. BoNT/B enzymatic domain, a BoNT/C1 enzymatic 0270 26. The method of 25, wherein the enkephalin is a domain, a BoNT/D enzymatic domain, a BoNT/E enzy Leu-enkephalin, a Met-enkephalin, a Met-enkephalin matic domain, a BoNT/F enzymatic domain, a BoNT/G MRGL or a Met-enkephalin MRF. enzymatic domain, a TeNT enzymatic domain, a BaNT 0271 27. The method of 25, wherein the enkephalin com enzymatic domain, or a BuNT enzymatic domain. prises SEQID NO:52, SEQID NO:53, SEQID NO:54 or 0284 40. The method of 23, wherein the exogenous pro SEQ ID NO:55. tease cleavage site is a plant papain cleavage site, an insect (0272. 28. The method of 25, wherein the BAM22 peptide papain cleavage site, a crustacian papain cleavage site, an is a BAM22 peptide (1-12), a BAM22 peptide (6-22), a enterokinase cleavage site, a human rhinovirus 3C protease BAM22 peptide (8-22) or a BAM22 peptide (1-22) cleavage site, a human enterovirus 3C protease cleavage (0273 29. The method of 25, wherein the BAM22 peptide site, a tobacco etch virus protease cleavage site, a Tobacco comprises amino acids 1-12, amino acids 6-22, amino Vein Mottling Virus cleavage site, a Subtilisin cleavage site, acids 8-22 or amino acids 1-22 of SEQID NO: 56; amino a hydroxylamine cleavage site, or a Caspase 3 cleavage acids 1-12, amino acids 6-22, amino acids 8-22 or amino site. acids 1-22 of SEQ ID NO: 57; amino acids 1-12, amino 0285) 41. The method of 23, wherein the urogenital-neu acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ rological disorder is urinary incontinence, overactive blad ID NO: 58; amino acids 1-12, amino acids 6-22, amino der, detrusor dysfunction, lower urinary tract dysfunction, acids 8-22 or amino acids 1-22 of SEQID NO. 59; amino urinary retention, urinary hesitancy, polyuria, nocturia, acids 1-12, amino acids 6-22, amino acids 8-22 or amino chronic urinary tract infection, an urogenital disorder asso acids 1-22 of SEQID NO: 60 or amino acids 1-12, amino ciated with a prostate disorder, an urogenital disorder asso acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ciated with a uterine disorder, or an urogenital disorder ID NO: 61 associated with a neurogenic dysfunction. 0274 30. The method of 25, wherein the endomorphin is 0286 42. The method of 41, wherein the urinary inconti an endomorphin-1 or an endomorphin-2. nence is an urge urinary incontinence, a stress urinary incontinence, an overflow urinary incontinence, a mixed (0275 31. The method of 25, wherein the endomorphin urinary incontinence, or a continuous urinary inconti comprises SEQID NO: 62 or SEQID NO: 63. CCC. (0276 32. The method of 25, wherein the endorphin an 0287 43. The method of 41, wherein the detrusor dysfunc endorphin-C, a neoendorphin-C., an endorphin-fi, a neoen tion is a detrusor overactivity, a detrusor instability, or a dorphin-for an endorphin-Y. detrusor-sphincter dyssynergia. (0277 33. The method of 25, wherein the endorphin com 0288 44. The method of 41, wherein the urogenital disor prises SEQID NO: 64, SEQID NO: 65, SEQID NO: 66, der associated with a prostate disorder is an urogenital SEQ ID NO: 67, SEQID NO: 68 or SEQID NO: 69. disorder associated with benign prostatic hyperplasia, an 0278 34. The method of 25, wherein the dynorphin is a urogenital disorder associated with prostatitis, or an uro dynorphin A, a dynorphin B (leumorphin) or a rimorphin. genital disorder associated with prostatodynia. (0279. 35. The method of 25, wherein the dynorphin com 0289 45. The method of 41, wherein the urogenital disor prises SEQID NO: 70, SEQID NO: 71, SEQID NO: 72, der associated with a neurogenic dysfunction is an urogeni SEQID NO: 73, SEQID NO: 74, SEQID NO: 75, SEQID tal disorder associated with Parkinson's Disease, an uro NO:76, SEQID NO: 77, SEQID NO: 78, SEQID NO: 79, genital disorder associated with multiple Sclerosis, an SEQID NO:80, SEQID NO:81, SEQID NO: 82, SEQID urogenital disorder associated with spina bifida, an uro NO: 83, SEQID NO:84, SEQID NO: 85, SEQID NO:86, genital disorder associated with transverse myelitis, an SEQID NO:87, SEQID NO: 88, SEQID NO: 89, SEQID urogenital disorder associated with stroke, an urogenital NO:90, SEQID NO:91, SEQID NO:92, SEQID NO:93, disorder associated with a spinal cord injury, an urogenital SEQID NO:94, SEQID NO:95, SEQID NO:96, SEQID disorder associated with a spasm reflex, an urogenital dis NO:97, SEQID NO: 98, SEQID NO: 99 or SEQID NO: order associated with a neurologic lesion of the spinal cord, 1OO. or an urogenital disorder associated with a neurologic 0280 36. The method of 25, wherein the nociceptin is a lesion of the brain. nociceptin RK, a nociceptin, a neuropeptide 1, a neuropep 0290 46. A use of a modified Clostridial toxin in the tide 2 or a neuropeptide 3. manufacturing a medicament for treating urogenital-neu US 2009/01171.57 A1 May 7, 2009 43
rological disorder in a mammal in need thereof, wherein NO:76, SEQID NO: 77, SEQID NO: 78, SEQID NO: 79, the modified Clostridial toxin comprising an opioid pep SEQID NO:80, SEQID NO:81, SEQID NO: 82, SEQID tide binding domain, a Clostridial toxin translocation NO: 83, SEQID NO: 84, SEQID NO: 85, SEQID NO: 86, domain and a Clostridial toxin enzymatic domain and SEQID NO: 87, SEQID NO: 88, SEQID NO: 89, SEQID wherein administration of a therapeutically effective NO:90, SEQID NO:91, SEQID NO:92, SEQID NO: 93, amount of the medicament to the mammal reduces a symp SEQID NO: 94, SEQID NO:95, SEQID NO:96, SEQID tom of the urogenital-neurological disorder, thereby treat NO:97, SEQID NO:98, SEQID NO: 99 or SEQID NO: ing the mammal. 1OO. 0291 47. The use of 46, wherein the modified Clostridial (0303 59. The method of 48, wherein the nociceptin is a toxin comprises a linear amino-to-carboxyl single nociceptin RK, a nociceptin, a neuropeptide 1, a neuropep polypeptide order of 1) the Clostridial toxin enzymatic tide 2 or a neuropeptide 3. domain, the Clostridial toxin translocation domain, the 0304) 60. The method of 48, wherein the nociceptin com opioid peptide binding domain, 2) the Clostridial toxin prises SEQ ID NO: 101, SEQID NO: 102, SEQ ID NO: enzymatic domain, the opioid peptide binding domain, the 103, SEQID NO: 104, SEQIDNO: 105, SEQIDNO: 106, Clostridial toxin translocation domain, 3) the opioid pep SEQ ID NO: 107, SEQ ID NO: 108, SEQID NO: 109 or tide binding domain, the Clostridial toxin translocation SEQID NO: 110. domain, and the Clostridial toxin enzymatic domain, 4) the 0305 61. The use of 46, wherein the Clostridial toxin opioid peptide binding domain, the Clostridial toxin enzy translocation domain is a BoNT/A translocation domain, a matic domain, the Clostridial toxin translocation domain, BoNT/B translocation domain, a BoNT/C1 translocation 5) the Clostridial toxin translocation domain, the domain, a BoNTVD translocation domain, a BoNT/E trans Clostridial toxin enzymatic domain and the opioid peptide location domain, a BoNT/F translocation domain, a binding domain, or 6) the Clostridial toxin translocation BoNT/G translocation domain, a TeNT translocation domain, the opioid peptide binding domain and the domain, a BaNT translocation domain, or a BuNT trans Clostridial toxin enzymatic domain. location domain. 0292) 48. The use of 46, wherein the opioid peptide bind ing domain is an enkephalin, a BAM22 peptide, an endo 0306 62. The use of 46, wherein the Clostridial toxin morphin, an endorphin, a dynorphin, a nociceptin or a enzymatic domain is a BoNT/A enzymatic domain, a hemorphin. BoNT/B enzymatic domain, a BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzy 0293 49. The method of 48, wherein the enkephalin is a matic domain, a BoNT/F enzymatic domain, a BoNT/G Leu-enkephalin, a Met-enkephalin, a Met-enkephalin enzymatic domain, a TeNT enzymatic domain, a BaNT MRGL or a Met-enkephalin MRF. enzymatic domain, or a BuNT enzymatic domain. 0294 50. The method of 48, wherein the enkephalin com prises SEQID NO:52, SEQID NO:53, SEQID NO:54 or 0307 63. The use of 46, wherein the urogenital-neurologi SEQ ID NO:55. cal disorder is urinary incontinence, overactive bladder, 0295) 51. The method of 48, wherein the BAM22 peptide detrusor dysfunction, lower urinary tract dysfunction, uri is a BAM22 peptide (1-12), a BAM22 peptide (6-22), a nary retention, urinary hesitancy, polyuria, nocturia, BAM22 peptide (8-22) or a BAM22 peptide (1-22) chronic urinary tract infection, an urogenital disorder asso 0296 52. The method of 48, wherein the BAM22 peptide ciated with a prostate disorder, an urogenital disorder asso comprises amino acids 1-12, amino acids 6-22, amino ciated with a uterine disorder, or an urogenital disorder acids 8-22 or amino acids 1-22 of SEQID NO: 56; amino associated with a neurogenic dysfunction. acids 1-12, amino acids 6-22, amino acids 8-22 or amino 0308) 64. The use of 63, wherein the urinary incontinence acids 1-22 of SEQ ID NO: 57; amino acids 1-12, amino is an urge urinary incontinence, a stress urinary inconti acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ nence, an overflow urinary incontinence, a mixed urinary ID NO: 58; amino acids 1-12, amino acids 6-22, amino incontinence, or a continuous urinary incontinence. acids 8-22 or amino acids 1-22 of SEQID NO. 59; amino (0309 65. The use of 63, wherein the detrusor dysfunction acids 1-12, amino acids 6-22, amino acids 8-22 or amino is a detrusor overactivity, a detrusor instability, or a detru acids 1-22 of SEQID NO: 60 or amino acids 1-12, amino sor-sphincter dyssynergia. acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ 0310) 66. The use of 63, wherein the urogenital disorder ID NO: 61 associated with a prostate disorder is an urogenital disorder 0297 53. The method of 48, wherein the endomorphin is associated with benign prostatic hyperplasia, an urogenital an endomorphin-1 or an endomorphin-2. disorder associated with prostatitis, or an urogenital disor 0298 54. The method of 48, wherein the endomorphin der associated with prostatodynia. comprises SEQID NO: 62 or SEQID NO: 63. 0311. 67. The use of 63, wherein the urogenital disorder 0299 55. The method of 48, wherein the endorphin an associated with a neurogenic dysfunction is an urogenital endorphin-C, a neoendorphin-C., an endorphin-fi, a neoen disorder associated with Parkinson's Disease, an urogeni dorphin-for an endorphin-Y. tal disorder associated with multiple Sclerosis, an urogeni 0300 56. The method of 48, wherein the endorphin com tal disorder associated with spina bifida, an urogenital dis prises SEQID NO: 64, SEQID NO: 65, SEQID NO: 66, order associated with transverse myelitis, an urogenital SEQ ID NO: 67, SEQID NO: 68 or SEQID NO: 69. disorder associated with stroke, an urogenital disorder 0301 57. The method of 48, wherein the dynorphin is a associated with a spinal cord injury, an urogenital disorder dynorphin A, a dynorphin B (leumorphin) or a rimorphin. associated with a spasm reflex, an urogenital disorder asso 0302) 58. The method of 48, wherein the dynorphin com ciated with a neurologic lesion of the spinal cord, or an prises SEQID NO: 70, SEQID NO: 71, SEQID NO: 72, urogenital disorder associated with a neurologic lesion of SEQID NO: 73, SEQID NO: 74, SEQID NO: 75, SEQID the brain. US 2009/01171.57 A1 May 7, 2009 44
0312 68. A use of a modified Clostridial toxin in the 0322 78. The method of 70, wherein the endorphin com manufacturing a medicament for treating urogenital-neu prises SEQID NO: 64, SEQID NO: 65, SEQID NO: 66, rological disorder in a mammal in need thereof, wherein SEQID NO: 67, SEQID NO: 68 or SEQID NO: 69. the modified Clostridial toxin comprising an opioid pep 0323 79. The method of 70, wherein the dynorphin is a tide binding domain, a Clostridial toxin translocation dynorphin A, a dynorphin B (leumorphin) or a rimorphin. domain and a Clostridial toxin enzymatic domain, and an 0324 80. The method of 70, wherein the dynorphin com exogenous protease cleavage site and wherein administra prises SEQID NO: 70, SEQID NO: 71, SEQID NO: 72, tion of a therapeutically effective amount of the medica SEQID NO: 73, SEQID NO: 74, SEQID NO: 75, SEQID ment to the mammal reduces a symptom of the urogenital NO:76, SEQID NO: 77, SEQID NO: 78, SEQID NO: 79, neurological disorder, thereby treating the mammal. SEQID NO:80, SEQID NO:81, SEQID NO: 82, SEQID NO: 83, SEQID NO: 84, SEQID NO: 85, SEQID NO: 86, 0313. 69. The use of 68, wherein the modified Clostridial SEQID NO: 87, SEQID NO: 88, SEQID NO: 89, SEQID toxin comprises a linear amino-to-carboxyl single NO:90, SEQID NO:91, SEQID NO:92, SEQID NO: 93, polypeptide order of 1) the Clostridial toxin enzymatic SEQID NO: 94, SEQID NO:95, SEQID NO:96, SEQID domain, the exogenous protease cleavage site, the NO:97, SEQID NO:98, SEQID NO: 99 or SEQID NO: Clostridial toxin translocation domain, the opioid peptide 1OO. binding domain, 2) the Clostridial toxin enzymatic 0325 81. The method of 70, wherein the nociceptin is a domain, the exogenous protease cleavage site, the opioid nociceptin RK, a nociceptin, a neuropeptide 1, a neuropep peptide binding domain, the Clostridial toxin translocation tide 2 or a neuropeptide 3. domain, 3) the opioid peptide binding domain, the 0326 82. The method of 70, wherein the nociceptin com Clostridial toxin translocation domain, the exogenous pro prises SEQ ID NO: 101, SEQID NO: 102, SEQ ID NO: tease cleavage site and the Clostridial toxin enzymatic 103, SEQID NO: 104, SEQIDNO: 105, SEQIDNO: 106, domain, 4) the opioid peptide binding domain, the SEQ ID NO: 107, SEQ ID NO: 108, SEQID NO: 109 or Clostridial toxin enzymatic domain, the exogenous pro SEQID NO: 110. tease cleavage site, the Clostridial toxin translocation 0327 83. The use of 68, wherein the Clostridial toxin domain, 5) the Clostridial toxin translocation domain, the translocation domain is a BoNT/A translocation domain, a exogenous protease cleavage site, the Clostridial toxin BoNT/B translocation domain, a BoNT/C1 translocation enzymatic domain and the opioid peptide binding domain, domain, a BoNTVD translocation domain, a BoNT/E trans or 6) the Clostridial toxin translocation domain, the exog location domain, a BoNT/F translocation domain, a enous protease cleavage site, the opioid peptide binding BoNT/G translocation domain, a TeNT translocation domain and the Clostridial toxin enzymatic domain. domain, a BaNT translocation domain, or a BuNT trans 0314. 70. The use of 69, wherein the opioid peptide bind location domain. ing domain is an enkephalin, a BAM22 peptide, an endo 0328 84. The use of 68, wherein the Clostridial toxin morphin, an endorphin, a dynorphin, a nociceptin or a enzymatic domain is a BoNT/A enzymatic domain, a hemorphin. BoNT/B enzymatic domain, a BoNT/C1 enzymatic 0315 71. The method of 70, wherein the enkephalin is a domain, a BoNT/D enzymatic domain, a BoNT/E enzy Leu-enkephalin, a Met-enkephalin, a Met-enkephalin matic domain, a BoNT/F enzymatic domain, a BoNT/G MRGL or a Met-enkephalin MRF. enzymatic domain, a TeNT enzymatic domain, a BaNT 0316 72. The method of 70, wherein the enkephalin com enzymatic domain, or a BuNT enzymatic domain. prises SEQID NO:52, SEQID NO:53, SEQID NO:54 or 0329 85. The use of 68, wherein the urogenital-neurologi SEQ ID NO:55. cal disorder is urinary incontinence, overactive bladder, detrusor dysfunction, lower urinary tract dysfunction, uri 0317. 73. The method of 70, wherein the BAM22 peptide nary retention, urinary hesitancy, polyuria, nocturia, is a BAM22 peptide (1-12), a BAM22 peptide (6-22), a chronic urinary tract infection, an urogenital disorder asso BAM22 peptide (8-22) or a BAM22 peptide (1-22) ciated with a prostate disorder, an urogenital disorder asso 0318 74. The method of 70, wherein the BAM22 peptide ciated with a uterine disorder, or an urogenital disorder comprises amino acids 1-12, amino acids 6-22, amino associated with a neurogenic dysfunction. acids 8-22 or amino acids 1-22 of SEQID NO: 56; amino 0330 86. The use of 85, wherein the urinary incontinence acids 1-12, amino acids 6-22, amino acids 8-22 or amino is an urge urinary incontinence, a stress urinary inconti acids 1-22 of SEQ ID NO: 57; amino acids 1-12, amino nence, an overflow urinary incontinence, a mixed urinary acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ incontinence, or a continuous urinary incontinence. ID NO: 58; amino acids 1-12, amino acids 6-22, amino 0331 87. The use of 85, wherein the detrusor dysfunction acids 8-22 or amino acids 1-22 of SEQID NO. 59; amino is a detrusor overactivity, a detrusor instability, or a detru acids 1-12, amino acids 6-22, amino acids 8-22 or amino sor-sphincter dyssynergia. acids 1-22 of SEQID NO: 60 or amino acids 1-12, amino 0332) 88. The use of 85, wherein the urogenital disorder acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ associated with a prostate disorder is an urogenital disorder ID NO: 61 associated with benign prostatic hyperplasia, an urogenital 0319 75. The method of 70, wherein the endomorphin is disorder associated with prostatitis, or an urogenital disor an endomorphin-1 or an endomorphin-2. der associated with prostatodynia. 0320) 76. The method of 70, wherein the endomorphin 0333 89. The use of 85, wherein the urogenital disorder comprises SEQID NO: 62 or SEQID NO: 63. associated with a neurogenic dysfunction is an urogenital 0321) 77. The method of 70, wherein the endorphin an disorder associated with Parkinson's Disease, an urogeni endorphin-C, a neoendorphin-C., an endorphin-fi, a neoen tal disorder associated with multiple Sclerosis, an urogeni dorphin-for an endorphin-Y. tal disorder associated with spina bifida, an urogenital dis US 2009/01171.57 A1 May 7, 2009 45
order associated with transverse myelitis, an urogenital administered into e.g., the detrusor, the bladder neck includ disorder associated with stroke, an urogenital disorder ing the external and internal urethral sphincters, the trigone, associated with a spinal cord injury, an urogenital disorder the bladder dome or other areas of the bladder wall, and/or associated with a spasm reflex, an urogenital disorder asso other areas Surrounding the bladder, Such as the urethra, ure ciated with a neurologic lesion of the spinal cord, or an ter, urogenital diaphragm, or lower pelvic muscles. The urogenital disorder associated with a neurologic lesion of patient's condition is monitored and after about 1-3 days from the brain. treatment, and the woman indicates there is improvement of 0334 90. A use of a modified Clostridial toxin for treating her ability to control the passage of urine, especially when she urogenital-neurological disorder in a mammal in need coughs, Sneezes, laughs or exercises. At one and three month thereof, the use comprising the step of administering to the check-ups, the woman indicates that she continues to have mammal in need thereofatherapeutically effective amount increased control over her ability to pass urine. This reduction of a composition, wherein the modified Clostridial toxin in a stressurinary incontinence symptom indicates Successful comprising an opioid peptide binding domain, a Clostridial treatment with the composition comprising a modified toxin translocation domain and a Clostridial toxin enzy Clostridial toxin. matic domain and wherein administration of the composi 0339. A 62 year old male complains of the inability to tion reduces a symptom of the urogenital-neurological dis control the passage of urine, experiencing a Sudden need to order, thereby treating the mammal. urinate. A physician diagnosis the patient with urge urinary 0335 91. A use of a modified Clostridial toxin for treating incontinence having a neurological component involving urogenital-neurological disorder in a mammal in need abnormal sensory neuron activity. The man is treated by thereof, the use comprising the step of administering to the injecting urethroscopically a composition comprising a mammal in need thereofatherapeutically effective amount modified Clostridial toxin as disclosed in the present specifi of a composition, wherein the modified Clostridial toxin cation. Depending on the location of abnormal sensory neu comprising an opioid peptide binding domain, a Clostridial ron activity, the toxin can be administered into e.g., the detru toxin translocation domain, a Clostridial toxin enzymatic Sor, the bladder neck including the external and internal domain, and an exogenous protease cleavage site, and urethral sphincters, the trigone, the bladder dome or other wherein administration of the composition reduces a areas of the bladder wall, and/or other areas surrounding the symptom of the urogenital-neurological disorder, thereby bladder, such as the urethra, ureter, urogenital diaphragm, lower pelvic muscles, prostate, bulbourethral gland, bulb, treating the mammal. crus or penis. The patient's condition is monitored and after 0336. The following examples are provided by way of about 1-3 days from treatment, and the man indicates there is describing specific embodiments without intending to limit improvement of his ability to control the passage of urine the scope of the invention in any way. because of a reduced Sudden need to urinate. At one and three month check-ups, the man indicates that he continues to have EXAMPLE1 increased control over his ability to pass urine. This reduction Treatment of Urinary Incontinence in an urge urinary incontinence symptom indicates Successful treatment with the composition comprising a modified 0337. A 69 year old female complains of the inability to Clostridial toxin. control the passage of urine. A physician diagnosis the patient 0340. A 58 year old male complains of the inability to with urinary incontinence having a neurological component control the passage of urine because of leakage that occurs. A involving abnormal sensory neuron activity. The woman is physician diagnosis the patient with overflow urinary incon treated by injecting urethroscopically a composition compris tinence having a neurological component involving abnormal ing a modified Clostridial toxin as disclosed in the present sensory neuron activity that is causing blockage. The man is specification. Depending on the location of abnormal sensory treated by injecting urethroscopically a composition compris neuron activity, the toxin can be administered into e.g., the ing a modified Clostridial toxin as disclosed in the present detrusor, the bladder neck including the external and internal specification. Depending on the location of abnormal sensory urethral sphincters, the trigone, the bladder dome or other neuron activity, the toxin can be administered into e.g., the areas of the bladder wall, and/or other areas surrounding the detrusor, the bladder neck including the external and internal bladder, Such as the urethra, ureter, urogenital diaphragm, or urethral sphincters, the trigone, the bladder dome or other lower pelvic muscles. The patient’s condition is monitored areas of the bladder wall, and/or other areas surrounding the and after about 1-3 days from treatment, and the woman bladder, such as the urethra, ureter, urogenital diaphragm, indicates there is improvement of her ability to control the lower pelvic muscles, prostate, bulbourethral gland, bulb, passage of urine. At one and three month check-ups, the crus or penis. The patient's condition is monitored and after woman indicates that she continues to have increased control about 1-3 days from treatment, and the man indicates there is over her ability to pass urine. This reduction in an urinary improvement of his ability to control the passage of urine incontinence symptom indicates Successful treatment with because of reduced leakage. At one and three month check the composition comprising a modified Clostridial toxin. ups, the man indicates that he continues to have increased 0338 A 72 year old female complains of the inability to control over his ability to pass urine. This reduction in an control the passage of urine, and leakage occurs especially overflow urinary incontinence symptom indicates Successful when she coughs, Sneezes, laughs or exercises. A physician treatment with the composition comprising a modified diagnosis the patient with stressurinary incontinence having Clostridial toxin. a neurological component involving abnormal sensory neu ron activity. The woman is treated by injecting urethroscopi EXAMPLE 2 cally a composition comprising a modified Clostridial toxin Treatment of Overactive Bladder as disclosed in the present specification. Depending on the 0341. A 58 year old male complains of increased urinary location of abnormal sensory neuron activity, the toxin can be urgency. A physician diagnosis the patient with overactive US 2009/01171.57 A1 May 7, 2009 46 bladder having a neurological component involving abnor neurological component involving abnormal sensory neuron mal sensory neuron activity. The man is treated by injecting activity. The man is treated by injecting urethroscopically a urethroscopically a composition comprising a modified composition comprising a modified Clostridial toxin as dis Clostridial toxin as disclosed in the present specification. closed in the present specification. Depending on the location Depending on the location of abnormal sensory neuron activ of abnormal sensory neuron activity, the toxin can be admin ity, the toxin can be administered into e.g., the detrusor, the istered into e.g., the detrusor, the bladder neck including the bladder neck including the external and internal urethral external and internal urethral sphincters, the trigone, the blad sphincters, the trigone, the bladder dome or other areas of the der dome or other areas of the bladder wall, and/or other areas bladder wall, and/or other areas surrounding the bladder, such Surrounding the bladder, Such as the urethra, ureter, urogeni as the urethra, ureter, urogenital diaphragm, lower pelvic tal diaphragm, lower pelvic muscles, prostate, bulbourethral muscles, prostate, bulbourethral gland, bulb, crus or penis. gland, bulb, crus or penis. The patient's condition is moni The patient's condition is monitored and after about 1-3 days tored and after about 1-3 days from treatment, and the man from treatment, and the man indicates that he has a reduced indicates that he has a reduced urgency to urinate. At one and urgency to urinate. At one and three month check-ups, the three month check-ups, the man indicates that he continues to man indicates that he continues to have a reduced urgency to have a reduced urgency to urinate. This reduction in an over urinate. This reduction in an overactive bladder symptom active bladder symptom indicates Successful treatment with indicates Successful treatment with the composition compris the composition comprising a modified Clostridial toxin. ing a modified Clostridial toxin. 0342. A 66 year old female complains of having to wake EXAMPLE 3 up several times during the night to urinate. A physician determines that this is nocturia and diagnosis the patient with Treatment of Detrusor Dysfunction overactive bladder having a neurological component involv ing abnormal sensory neuron activity. The woman is treated 0345. A 44 year old female complains of uncontrollable by injecting urethroscopically a composition comprising a bladder contractions. A physician determines that this is unin modified Clostridial toxin as disclosed in the present specifi hibitable bladder contractions and diagnosis the patient with cation. Depending on the location of abnormal sensory neu a detrusor dysfunction having a neurological component ron activity, the toxin can be administered into e.g., the detru involving abnormal sensory neuron activity. The woman is Sor, the bladder neck including the external and internal treated by injecting urethroscopically a composition compris urethral sphincters, the trigone, the bladder dome or other ing a modified Clostridial toxin as disclosed in the present areas of the bladder wall, and/or other areas surrounding the specification. Depending on the location of abnormal sensory bladder, Such as the urethra, ureter, urogenital diaphragm, or neuron activity, the toxin can be administered into e.g., the lower pelvic muscles. The patient’s condition is monitored detrusor, the bladder neck including the external and internal and after about 1-3 days from treatment, and the woman urethral sphincters, the trigone, the bladder dome or other indicates that she has a reduced need to wake up several times areas of the bladder wall, and/or other areas surrounding the during the night to urinate. At one and three month check-ups, bladder, Such as the urethra, ureter, urogenital diaphragm, or the woman indicates that she continues to have a reduced need lower pelvic muscles. The patient’s condition is monitored to wake up several times during the night to urinate. This and after about 1-3 days from treatment, and the woman reduction in an overactive bladder symptom indicates suc indicates that there is a reduction in uncontrollable bladder cessful treatment with the composition comprising a modi contractions. At one and three month check-ups, the woman fied Clostridial toxin. indicates that she continues to have a reduction in uncontrol 0343 A 47 year old female complains of having to urinate lable bladder contractions. This reduction in a detrusor dys several times a day. A physician determines that this is poly function symptom indicates Successful treatment with the uria and diagnosis the patient with overactive bladder having composition comprising a modified Clostridial toxin. a neurological component involving abnormal sensory neu 0346. In an alternative scenario, the physician determines ron activity. The woman is treated by injecting urethroscopi that this is uninhibitable bladder contractions and diagnosis cally a composition comprising a modified Clostridial toxin the patient with detrusor overactivity having a neurological as disclosed in the present specification. Depending on the component involving abnormal sensory neuron activity. The location of abnormal sensory neuron activity, the toxin can be woman is treated by injecting urethroscopically a composi administered into e.g., the detrusor, the bladder neck includ tion comprising a modified Clostridial toxin as disclosed in ing the external and internal urethral sphincters, the trigone, the present specification. Depending on the location of abnor the bladder dome or other areas of the bladder wall, and/or mal sensory neuron activity, the toxin can be administered other areas Surrounding the bladder, Such as the urethra, ure into e.g., the detrusor, the bladder neck including the external ter, urogenital diaphragm, or lower pelvic muscles. The and internal urethral sphincters, the trigone, the bladder dome patient's condition is monitored and after about 1-3 days from or other areas of the bladder wall, and/or other areas Sur treatment, and the woman indicates that she has a reduced rounding the bladder, such as the urethra, ureter, urogenital need to urinate during the day. At one and three month check diaphragm, or lower pelvic muscles. The patient's condition ups, the woman indicates that she continues to have a reduced is monitored and after about 1-3 days from treatment, and the need urinate during the day. This reduction in an overactive woman indicates that there is a reduction in uncontrollable bladder symptom indicates successful treatment with the bladder contractions. At one and three month check-ups, the composition comprising a modified Clostridial toxin. woman indicates that she continues to have a reduction in 0344. A 67 year old male complains of the inability to uncontrollable bladder contractions. This reduction in a control the passage of urine because of a Sudden need to detrusor overactivity symptom indicates Successful treatment urinate. A physician determines that this is urge incontinence with the composition comprising a modified Clostridial and diagnosis the patient with overactive bladder having a toxin. US 2009/01171.57 A1 May 7, 2009 47
0347 In another alternative scenario, the physician deter involving abnormal sensory neuron activity. The woman is mines that this is uninhibitable bladder contractions and diag treated by injecting urethroscopically a composition compris nosis the patient with detrusor instability having a neurologi ing a modified Clostridial toxin as disclosed in the present cal component involving abnormal sensory neuron activity. specification. Depending on the location of abnormal sensory The woman is treated by injecting urethroscopically a com neuron activity, the toxin can be administered into e.g., the position comprising a modified Clostridial toxin as disclosed detrusor, the bladder neck including the external and internal in the present specification. Depending on the location of urethral sphincters, the trigone, the bladder dome or other abnormal sensory neuron activity, the toxin can be adminis areas of the bladder wall, and/or other areas surrounding the tered into e.g., the detrusor, the bladder neck including the bladder, Such as the urethra, ureter, urogenital diaphragm, or external and internal urethral sphincters, the trigone, the blad lower pelvic muscles. The patient’s condition is monitored der dome or other areas of the bladder wall, and/or other areas and after about 1-3 days from treatment, and the woman Surrounding the bladder. Such as the urethra, ureter, urogeni indicates that there is a reduction in the urgency to urinate. At tal diaphragm, or lower pelvic muscles. The patient's condi one and three month check-ups, the woman indicates that she tion is monitored and after about 1-3 days from treatment, and continues to have a reduction in the urgency to urinate. This the woman indicates that there is a reduction in uncontrollable reductionina detrusor instability symptom indicates Success bladder contractions. At one and three month check-ups, the ful treatment with the composition comprising a modified woman indicates that she continues to have a reduction in Clostridial toxin. uncontrollable bladder contractions. This reduction in a 0351. A 59 year old male complains of having to urinate detrusor instability symptom indicates Successful treatment all the time. A physician determines that this is urinary fre with the composition comprising a modified Clostridial quency and diagnosis the patient with a detrusor dysfunction toxin. having a neurological component involving abnormal sen 0348. A 50 year old female complains of an urgency to sory neuron activity. The man is treated by injecting urethro urinate. A physician determines that this is urinary urgency scopically a composition comprising a modified Clostridial and diagnosis the patient with a detrusor dysfunction having toxin as disclosed in the present specification. Depending on a neurological component involving abnormal sensory neu the location of abnormal sensory neuron activity, the toxin ron activity. The woman is treated by injecting urethroscopi can be administered into e.g., the detrusor, the bladder neck cally a composition comprising a modified Clostridial toxin including the external and internal urethral sphincters, the as disclosed in the present specification. Depending on the trigone, the bladder dome or other areas of the bladder wall, location of abnormal sensory neuron activity, the toxin can be and/or other areas surrounding the bladder, such as the ure administered into e.g., the detrusor, the bladder neck includ thra, ureter, urogenital diaphragm, lower pelvic muscles, ing the external and internal urethral sphincters, the trigone, prostate, bulbourethral gland, bulb, crus or penis. The the bladder dome or other areas of the bladder wall, and/or patient's condition is monitored and after about 1-3 days from other areas Surrounding the bladder, Such as the urethra, ure treatment, and the man indicates that there is a reduction in the ter, urogenital diaphragm, or lower pelvic muscles. The need to urinate all the time. At one and three month check patient's condition is monitored and after about 1-3 days from ups, the man indicates that he continues to have a reduction in treatment, and the woman indicates that there is a reduction in the need to urinate all the time. This reduction in a detrusor the urgency to urinate. At one and three month check-ups, the dysfunction symptom indicates Successful treatment with the woman indicates that she continues to have a reduction in the composition comprising a modified Clostridial toxin. urgency to urinate. This reduction in a detrusor dysfunction 0352. In an alternative scenario, the physician determines symptom indicates Successful treatment with the composi that this is urinary frequency and diagnosis the patient with tion comprising a modified Clostridial toxin. detrusor overactivity having a neurological component 0349. In an alternative scenario, the physician determines involving abnormal sensory neuron activity. The man is that this is urinary urgency and diagnosis the patient with treated by injecting urethroscopically a composition compris detrusor overactivity having a neurological component ing a modified Clostridial toxin as disclosed in the present involving abnormal sensory neuron activity. The woman is specification. Depending on the location of abnormal sensory treated by injecting urethroscopically a composition compris neuron activity, the toxin can be administered into e.g., the ing a modified Clostridial toxin as disclosed in the present detrusor, the bladder neck including the external and internal specification. Depending on the location of abnormal sensory urethral sphincters, the trigone, the bladder dome or other neuron activity, the toxin can be administered into e.g., the areas of the bladder wall, and/or other areas surrounding the detrusor, the bladder neck including the external and internal bladder, Such as the urethra, ureter, urogenital diaphragm, or urethral sphincters, the trigone, the bladder dome or other lower pelvic muscles. The patient’s condition is monitored areas of the bladder wall, and/or other areas surrounding the and after about 1-3 days from treatment, and the man indi bladder, Such as the urethra, ureter, urogenital diaphragm, or cates that there is a reduction in the need to urinate all the lower pelvic muscles. The patient’s condition is monitored time. At one and three month check-ups, the man indicates and after about 1-3 days from treatment, and the woman that he continues to have a reduction in the need to urinate all indicates that there is a reduction in the urgency to urinate. At the time. This reduction in a detrusor overactivity symptom one and three month check-ups, the woman indicates that she indicates Successful treatment with the composition compris continues to have a reduction in the urgency to urinate. This ing a modified Clostridial toxin. reduction in a detrusor overactivity symptom indicates suc 0353. In another alternative scenario, the physician deter cessful treatment with the composition comprising a modi mines that this is urinary frequency and diagnosis the patient fied Clostridial toxin. with detrusor instability having a neurological component 0350. In another alternative scenario, the physician deter involving abnormal sensory neuron activity. The man is mines that this is urinary urgency and diagnosis the patient treated by injecting urethroscopically a composition compris with detrusor instability having a neurological component ing a modified Clostridial toxin as disclosed in the present US 2009/01171.57 A1 May 7, 2009 48 specification. Depending on the location of abnormal sensory urethral sphincters, the trigone, the bladder dome or other neuron activity, the toxin can be administered into e.g., the areas of the bladder wall, and/or other areas surrounding the detrusor, the bladder neck including the external and internal bladder, Such as the urethra, ureter, urogenital diaphragm, or urethral sphincters, the trigone, the bladder dome or other lower pelvic muscles. The patient’s condition is monitored areas of the bladder wall, and/or other areas surrounding the and after about 1-3 days from treatment, and the man indi bladder, Such as the urethra, ureter, urogenital diaphragm, or cates that there is a reduction in the involuntary loss of urine. lower pelvic muscles. The patient’s condition is monitored At one and three month check-ups, the man indicates that he and after about 1-3 days from treatment, and the man indi continues to have a reduction in the involuntary loss of urine. cates that there is a reduction in the need to urinate all the This reduction in a detrusor instability symptom indicates time. At one and three month check-ups, the man indicates Successful treatment with the composition comprising a that he continues to have a reduction in the need to urinate all modified Clostridial toxin. the time. This reduction in a detrusor instability symptom 0357. A 63 year old male complains of having to wake up indicates Successful treatment with the composition compris several times during the night to urinate. A physician deter ing a modified Clostridial toxin. mines that this is nocturia and diagnosis the patient with a 0354 A74 year old male complains of the involuntary loss detrusor dysfunction having a neurological component of urine. A physician determines that this is enuresis and involving abnormal sensory neuron activity. The man is diagnosis the patient with a detrusor dysfunction having a treated by injecting urethroscopically a composition compris neurological component involving abnormal sensory neuron ing a modified Clostridial toxin as disclosed in the present activity. The man is treated by injecting urethroscopically a specification. Depending on the location of abnormal sensory composition comprising a modified Clostridial toxin as dis neuron activity, the toxin can be administered into e.g., the closed in the present specification. Depending on the location detrusor, the bladder neck including the external and internal of abnormal sensory neuron activity, the toxin can be admin urethral sphincters, the trigone, the bladder dome or other istered into e.g., the detrusor, the bladder neck including the areas of the bladder wall, and/or other areas surrounding the external and internal urethral sphincters, the trigone, the blad bladder, such as the urethra, ureter, urogenital diaphragm, der dome or other areas of the bladder wall, and/or other areas lower pelvic muscles, prostate, bulbourethral gland, bulb, Surrounding the bladder. Such as the urethra, ureter, urogeni crus or penis. The patient's condition is monitored and after tal diaphragm, lower pelvic muscles, prostate, bulbourethral about 1-3 days from treatment, and the man indicates that gland, bulb, crus or penis. The patient's condition is moni there is a reduction in need to wake up several times during tored and after about 1-3 days from treatment, and the man the night to urinate. At one and three month check-ups, the indicates that there is a reduction in the involuntary loss of man indicates that he continues to have a reduction in need to urine. At one and three month check-ups, the man indicates wake up several times during the night to urinate. This reduc that he continues to have a reduction in the involuntary loss of tion in a detrusor dysfunction symptom indicates Successful urine. This reduction in a detrusor dysfunction symptom indi treatment with the composition comprising a modified cates successful treatment with the composition comprising a Clostridial toxin. modified Clostridial toxin. 0358. In an alternative scenario, the physician determines 0355. In an alternative scenario, the physician determines that this is nocturia and diagnosis the patient with detrusor that this is enuresis and diagnosis the patient with detrusor overactivity having a neurological component involving overactivity having a neurological component involving abnormal sensory neuron activity. The man is treated by abnormal sensory neuron activity. The man is treated by injecting urethroscopically a composition comprising a injecting urethroscopically a composition comprising a modified Clostridial toxin as disclosed in the present specifi modified Clostridial toxin as disclosed in the present specifi cation. Depending on the location of abnormal sensory neu cation. Depending on the location of abnormal sensory neu ron activity, the toxin can be administered into e.g., the detru ron activity, the toxin can be administered into e.g., the detru Sor, the bladder neck including the external and internal Sor, the bladder neck including the external and internal urethral sphincters, the trigone, the bladder dome or other urethral sphincters, the trigone, the bladder dome or other areas of the bladder wall, and/or other areas surrounding the areas of the bladder wall, and/or other areas surrounding the bladder, Such as the urethra, ureter, urogenital diaphragm, or bladder, Such as the urethra, ureter, urogenital diaphragm, or lower pelvic muscles. The patient’s condition is monitored lower pelvic muscles. The patient’s condition is monitored and after about 1-3 days from treatment, and the man indi and after about 1-3 days from treatment, and the man indi cates that there is a reduction in need to wake up several times cates that there is a reduction in the involuntary loss of urine. during the night to urinate. At one and three month check-ups, At one and three month check-ups, the man indicates that he the man indicates that he continues to have a reduction in need continues to have a reduction in the involuntary loss of urine. to wake up several times during the night to urinate. This This reduction in a detrusor overactivity symptom indicates reduction in a detrusor overactivity symptom indicates Suc Successful treatment with the composition comprising a cessful treatment with the composition comprising a modi modified Clostridial toxin. fied Clostridial toxin. 0356. In another alternative scenario, the physician deter 0359. In another alternative scenario, the physician deter mines that this is enuresis and diagnosis the patient with mines that this is nocturia and diagnosis the patient with detrusor instability having a neurological component involv detrusor instability having a neurological component involv ing abnormal sensory neuron activity. The man is treated by ing abnormal sensory neuron activity. The man is treated by injecting urethroscopically a composition comprising a injecting urethroscopically a composition comprising a modified Clostridial toxin as disclosed in the present specifi modified Clostridial toxin as disclosed in the present specifi cation. Depending on the location of abnormal sensory neu cation. Depending on the location of abnormal sensory neu ron activity, the toxin can be administered into e.g., the detru ron activity, the toxin can be administered into e.g., the detru Sor, the bladder neck including the external and internal Sor, the bladder neck including the external and internal US 2009/01171.57 A1 May 7, 2009 49 urethral sphincters, the trigone, the bladder dome or other bladder, Such as the urethra, ureter, urogenital diaphragm, or areas of the bladder wall, and/or other areas surrounding the lower pelvic muscles. The patient’s condition is monitored bladder, Such as the urethra, ureter, urogenital diaphragm, or and after about 1-3 days from treatment, and the woman lower pelvic muscles. The patient’s condition is monitored indicates that there is a reduction in the need to urinate several and after about 1-3 days from treatment, and the man indi times a day. At one and three month check-ups, the woman cates that there is a reduction in need to wake up several times indicates that she continues to have a reduction in the need to during the night to urinate. At one and three month check-ups, urinate several times a day. This reduction in a detrusor insta the man indicates that he continues to have a reduction in need bility symptom indicates successful treatment with the com to wake up several times during the night to urinate. This position comprising a modified Clostridial toxin. reduction in a detrusor instability symptom indicates Success 0363 A 65 year old female complains of the inability to ful treatment with the composition comprising a modified control the passage of urine. A physician determines that this Clostridial toxin. is urinary incontinence and diagnosis the patient with a detru 0360. A 61 year old female complains of having to urinate Sor dysfunction having a neurological component involving several times a day. A physician determines that this is poly abnormal sensory neuron activity. The woman is treated by uria and diagnosis the patient with a detrusor dysfunction injecting urethroscopically a composition comprising a having a neurological component involving abnormal sen modified Clostridial toxin as disclosed in the present specifi sory neuron activity. The woman is treated by injecting ure cation. Depending on the location of abnormal sensory neu throscopically a composition comprising a modified ron activity, the toxin can be administered into e.g., the detru Clostridial toxin as disclosed in the present specification. Sor, the bladder neck including the external and internal Depending on the location of abnormal sensory neuron activ urethral sphincters, the trigone, the bladder dome or other ity, the toxin can be administered into e.g., the detrusor, the areas of the bladder wall, and/or other areas surrounding the bladder neck including the external and internal urethral bladder, Such as the urethra, ureter, urogenital diaphragm, or sphincters, the trigone, the bladder dome or other areas of the lower pelvic muscles. The patient’s condition is monitored bladder wall, and/or other areas surrounding the bladder, such and after about 1-3 days from the treatment, and the woman as the urethra, ureter, urogenital diaphragm, or lower pelvic indicates there is improvement of her ability to control the muscles. The patient's condition is monitored and after about passage of urine. At one and three month check-ups, the 1-3 days from treatment, and the woman indicates that there woman indicates that she continues to have an improved is a reduction in the need to urinate several times a day. At one ability to control the passage of urine since the treatment. This and three month check-ups, the woman indicates that she reduction in a detrusor dysfunction symptom indicates suc continues to have a reduction in the need to urinate several cessful treatment with the composition comprising a modi times a day. This reduction in a detrusor dysfunction symp fied Clostridial toxin. tom indicates Successful treatment with the composition 0364. In an alternative scenario, the physician determines comprising a modified Clostridial toxin. that this is urinary incontinence and diagnosis the patient with 0361. In an alternative scenario, the physician determines detrusor overactivity having a neurological component that this is polyuria and diagnosis the patient with detrusor involving abnormal sensory neuron activity. The woman is overactivity having a neurological component involving treated by injecting urethroscopically a composition compris abnormal sensory neuron activity. The woman is treated by ing a modified Clostridial toxin as disclosed in the present injecting urethroscopically a composition comprising a specification. Depending on the location of abnormal sensory modified Clostridial toxin as disclosed in the present specifi neuron activity, the toxin can be administered into e.g., the cation. Depending on the location of abnormal sensory neu detrusor, the bladder neck including the external and internal ron activity, the toxin can be administered into e.g., the detru urethral sphincters, the trigone, the bladder dome or other Sor, the bladder neck including the external and internal areas of the bladder wall, and/or other areas surrounding the urethral sphincters, the trigone, the bladder dome or other bladder, Such as the urethra, ureter, urogenital diaphragm, or areas of the bladder wall, and/or other areas surrounding the lower pelvic muscles. The patient’s condition is monitored bladder, Such as the urethra, ureter, urogenital diaphragm, or and after about 1-3 days from the treatment, and the woman lower pelvic muscles. The patient’s condition is monitored indicates there is improvement of her ability to control the and after about 1-3 days from treatment, and the woman passage of urine. At one and three month check-ups, the indicates that there is a reduction in the need to urinate several woman indicates that she continues to have an improved times a day. At one and three month check-ups, the woman ability to control the passage of urine since the treatment. This indicates that she continues to have a reduction in the need to reduction in a detrusor overactivity symptom indicates Suc urinate several times a day. This reduction in a detrusor over cessful treatment with the composition comprising a modi activity symptom indicates successful treatment with the fied Clostridial toxin. composition comprising a modified Clostridial toxin. 0365. In another alternative scenario, the physician deter 0362. In another alternative scenario, the physician deter mines that this is urinary incontinence and diagnosis the mines that this is polyuria and diagnosis the patient with patient with detrusor instability having a neurological com detrusor instability having a neurological component involv ponent involving abnormal sensory neuron activity. The ing abnormal sensory neuron activity. The woman is treated woman is treated by injecting urethroscopically a composi by injecting urethroscopically a composition comprising a tion comprising a modified Clostridial toxin as disclosed in modified Clostridial toxin as disclosed in the present specifi the present specification. Depending on the location of abnor cation. Depending on the location of abnormal sensory neu mal sensory neuron activity, the toxin can be administered ron activity, the toxin can be administered into e.g., the detru into e.g., the detrusor, the bladder neck including the external Sor, the bladder neck including the external and internal and internal urethral sphincters, the trigone, the bladder dome urethral sphincters, the trigone, the bladder dome or other or other areas of the bladder wall, and/or other areas Sur areas of the bladder wall, and/or other areas surrounding the rounding the bladder, such as the urethra, ureter, urogenital US 2009/01171.57 A1 May 7, 2009 50 diaphragm, or lower pelvic muscles. The patient's condition treatment, and the man indicates that there is a reduction in is monitored and after about 1-3 days from the treatment, and bladder pressure. At one and three month check-ups, the man the woman indicates there is improvement of her ability to indicates that he continues to have a reduced bladder pressure control the passage of urine. At one and three month check since the treatment. This reduction in a detrusor dysfunction ups, the woman indicates that she continues to have an symptom indicates Successful treatment with the composi improved ability to control the passage of urine since the tion comprising a modified Clostridial toxin. treatment. This reduction in a detrusor instability symptom 0369. In an alternative scenario, the physician determines indicates Successful treatment with the composition compris that this is raised detrusor pressure and diagnosis the patient ing a modified Clostridial toxin. with a detrusor-sphincter dyssynergia having a neurological 0366 A55 year old female complains of an interruption of component involving abnormal sensory neuron activity. The urine flow when she urinates. A physician diagnosis the man is treated by injecting urethroscopically a composition patient with a detrusor dysfunction having a neurological comprising a modified Clostridial toxin as disclosed in the component involving abnormal sensory neuron activity. The present specification. Depending on the location of abnormal woman is treated by injecting urethroscopically a composi sensory neuron activity, the toxin can be administered into tion comprising a modified Clostridial toxin as disclosed in e.g., the detrusor, the bladder neck including the external and the present specification. Depending on the location of abnor internal urethral sphincters, the trigone, the bladder dome or mal sensory neuron activity, the toxin can be administered other areas of the bladder wall, and/or other areas surrounding into e.g., the detrusor, the bladder neck including the external the bladder, Such as the urethra, ureter, urogenital diaphragm, and internal urethral sphincters, the trigone, the bladder dome or lower pelvic muscles. The patient's condition is monitored or other areas of the bladder wall, and/or other areas Sur and after about 1-3 days from treatment, and the man indi rounding the bladder, Such as the urethra, ureter, urogenital cates that there is a reduction in bladder pressure. At one and diaphragm, or lower pelvic muscles. The patient's condition three month check-ups, the man indicates that he continues to is monitored and after about 1-3 days from treatment, and the have a reduced bladder pressure since the treatment. This woman indicates that there is a reduction in urine flow inter reduction in a detrusor-sphincter dyssynergia symptom indi ruption. At one and three month check-ups, the woman indi cates successful treatment with the composition comprising a cates that she continues to have a reduced urine flow inter modified Clostridial toxin. ruption since the treatment. This reduction in a detrusor 0370. A 75 year old male complains of the inability to dysfunction symptom indicates successful treatment with the urinate. A physician determines that this is urinary retention composition comprising a modified Clostridial toxin. and diagnosis the patient with a detrusor dysfunction having 0367. In an alternative scenario, the physician diagnosis a neurological component involving abnormal sensory neu the patient with a detrusor-sphincter dyssynergia having a ron activity. The man is treated by injecting urethroscopically neurological component involving abnormal sensory neuron a composition comprising a modified Clostridial toxin as activity. The woman is treated by injecting urethroscopically disclosed in the present specification. Depending on the loca a composition comprising a modified Clostridial toxin as tion of abnormal sensory neuron activity, the toxin can be disclosed in the present specification. Depending on the loca administered into e.g., the detrusor, the bladder neck includ tion of abnormal sensory neuron activity, the toxin can be ing the external and internal urethral sphincters, the trigone, administered into e.g., the detrusor, the bladder neck includ the bladder dome or other areas of the bladder wall, and/or ing the external and internal urethral sphincters, the trigone, other areas Surrounding the bladder, Such as the urethra, ure the bladder dome or other areas of the bladder wall, and/or ter, urogenital diaphragm, lower pelvic muscles, prostate, other areas Surrounding the bladder, Such as the urethra, ure bulbourethral gland, bulb, crus or penis. The patient's condi ter, urogenital diaphragm, or lower pelvic muscles. The tion is monitored and after about 1-3 days from treatment, and patient's condition is monitored and after about 1-3 days from the man indicates that he has regained the ability tourinate. At treatment, and the woman indicates that there is a reduction in one and three month check-ups, the man indicates that he urine flow interruption. At one and three month check-ups, continues to have the ability to urinate. This reduction in a the woman indicates that she continues to have a reduced detrusor dysfunction symptom indicates Successful treatment urine flow interruption since the treatment. This reduction in with the composition comprising a modified Clostridial a detrusor-sphincter dyssynergia symptom indicates Success toxin. ful treatment with the composition comprising a modified 0371. In an alternative scenario, the physician determines Clostridial toxin. that this is urinary retention and diagnosis the patient with a 0368. A 53 year old male complains of increased bladder detrusor-sphincter dyssynergia having a neurological compo pressure. A physician determines that this is raised detrusor nent involving abnormal sensory neuron activity. The man is pressure and diagnosis the patient with a detrusor dysfunction treated by injecting urethroscopically a composition compris having a neurological component involving abnormal sen ing a modified Clostridial toxin as disclosed in the present sory neuron activity. The man is treated by injecting urethro specification. Depending on the location of abnormal sensory scopically a composition comprising a modified Clostridial neuron activity, the toxin can be administered into e.g., the toxin as disclosed in the present specification. Depending on detrusor, the bladder neck including the external and internal the location of abnormal sensory neuron activity, the toxin urethral sphincters, the trigone, the bladder dome or other can be administered into e.g., the detrusor, the bladder neck areas of the bladder wall, and/or other areas surrounding the including the external and internal urethral sphincters, the bladder, Such as the urethra, ureter, urogenital diaphragm, or trigone, the bladder dome or other areas of the bladder wall, lower pelvic muscles. The patient’s condition is monitored and/or other areas Surrounding the bladder, such as the ure and after about 1-3 days from treatment, and the man indi thra, ureter, urogenital diaphragm, lower pelvic muscles, cates that he has regained the ability to urinate. At one and prostate, bulbourethral gland, bulb, crus or penis. The three month check-ups, the man indicates that he continues to patient's condition is monitored and after about 1-3 days from have the ability to urinate. This reduction in a detrusor US 2009/01171.57 A1 May 7, 2009
sphincter dyssynergia symptom indicates successful treat tion, a physician would treat the patient as indicated above ment with the composition comprising a modified Clostridial and there would be a reduction in the lower urinary tract toxin. dysfunction Voiding symptom. 0374. A 77 year old male complains of urine dribbling EXAMPLE 4 after he finishes urinating. A physician determines that this is a urine post-micturition problem and diagnosis the patient Treatment of Lower Urinary Tract Dysfunction with a lower urinary tract dysfunction having a neurological 0372. A 69 year old male complains of the need to urinate component involving abnormal sensory neuron activity. The Suddenly. A physician determines that this is a urine storage man is treated by injecting urethroscopically a composition problem and diagnosis the patient with a lower urinary tract comprising a modified Clostridial toxin as disclosed in the dysfunction having a neurological component involving present specification. Depending on the location of abnormal abnormal sensory neuron activity. The man is treated by sensory neuron activity, the toxin can be administered into injecting urethroscopically a composition comprising a e.g., the detrusor, the bladder neck including the external and modified Clostridial toxin as disclosed in the present specifi internal urethral sphincters, the trigone, the bladder dome or cation. Depending on the location of abnormal sensory neu other areas of the bladder wall, and/or other areas surrounding ron activity, the toxin can be administered into e.g., the detru the bladder, Such as the urethra, ureter, urogenital diaphragm, Sor, the bladder neck including the external and internal lower pelvic muscles, prostate, bulbourethral gland, bulb, urethral sphincters, the trigone, the bladder dome or other crus or penis. The patient's condition is monitored and after areas of the bladder wall, and/or other areas surrounding the about 1-3 days from treatment, and the man indicates that bladder, Such as the urethra, ureter, urogenital diaphragm, there is a reduction in urine dribbling after he finishes urinat lower pelvic muscles, prostate, bulbourethral gland, bulb, ing. At one and three month check-ups, the man indicates that crus or penis. The patient's condition is monitored and after he still experiences reduced dribbling after he finishes urinat about 1-3 days from treatment, and the man indicates that ing. This reduction in a lower urinary tract dysfunction indi there is a reduction in the Sudden need to urinate. At one and cates successful treatment with the composition comprising a three month check-ups, the man indicates that he still expe modified Clostridial toxin. In similar scenarios the patient riences a reduced need to urinate. This reduction in a lower could have complained of other post-micturition symptoms urinary tract dysfunction indicates Successful treatment with of lower urinary tract dysfunction Such as, e.g., sensation of the composition comprising a modified Clostridial toxin. In incomplete emptying. In each case, after diagnosis of lower similar scenarios the patient could have complained of other urinary tract dysfunction, a physician would treat the patient storage symptoms of lower urinary tract dysfunction Such as, as indicated above and there would beareduction in the lower e.g., urinary frequency, enuresis, polyuria, nocturia increased urinary tract dysfunction post-micturition symptom. bladder sensation, decreased bladder sensation, absent blad der sensation, non-specific bladder sensation, and/or urinary EXAMPLE 5 incontinence. In each case, after diagnosis of lower urinary Treatment of Urinary Retention tract dysfunction, a physician would treat the patient as indi cated above and there would be a reduction in the lower 0375. A 79 year old female complains that she cannot urinary tract dysfunction storage symptom. urinate. A physician diagnosis the patient with urinary reten 0373 A 70 year old male complains of having difficulty tion having a neurological component involving abnormal urinating and having to strain in order to urinate. A physician sensory neuron activity. The woman is treated by injecting determines that this is a urine Voiding problem and diagnosis urethroscopically a composition comprising a modified the patient with a lower urinary tract dysfunction having a Clostridial toxin as disclosed in the present specification. neurological component involving abnormal sensory neuron Depending on the location of abnormal sensory neuron activ activity. The man is treated by injecting urethroscopically a ity, the toxin can be administered into e.g., the detrusor, the composition comprising a modified Clostridial toxin as dis bladder neck including the external and internal urethral closed in the present specification. Depending on the location sphincters, the trigone, the bladder dome or other areas of the of abnormal sensory neuron activity, the toxin can be admin bladder wall, and/or other areas surrounding the bladder, such istered into e.g., the detrusor, the bladder neck including the as the urethra, ureter, urogenital diaphragm, or lower pelvic external and internal urethral sphincters, the trigone, the blad muscles. The patient's condition is monitored and after about der dome or other areas of the bladder wall, and/or other areas 1-3 days from treatment, and the woman indicates that she has Surrounding the bladder. Such as the urethra, ureter, urogeni regained the ability to urinate. At one and three month check tal diaphragm, lower pelvic muscles, prostate, bulbourethral ups, the woman indicates that she still continues to have gland, bulb, crus or penis. The patient's condition is moni control over her ability to urinate. This reduction in a urinary tored and after about 1-3 days from treatment, and the man retention symptom indicates successful treatment with the indicates that it is easier to urinate and he does not have to composition comprising a modified Clostridial toxin. strain as much in order to urinate. At one and three month check-ups, the man indicates that he still experiences an EXAMPLE 6 easier time to urinate. This reduction in a lower urinary tract Treatment of Urinary Hesitancy dysfunction indicates successful treatment with the compo sition comprising a modified Clostridial toxin. In similar 0376. A 78 year old male complains that he has difficulty scenarios the patient could have complained of other voiding starting and/or maintaining his ability to urinate. A physician symptoms of lower urinary tract dysfunction such as, e.g., diagnosis the patient with urinary hesitancy having a neuro reducedurine flow, splitting or spraying of urine, intermittent logical component involving abnormal sensory neuron activ urine flow, urinary hesitancy, and/or terminal dribble of urine. ity. The man is treated by injecting urethroscopically a com In each case, after diagnosis of lower urinary tract dysfunc position comprising a modified Clostridial toxin as disclosed US 2009/01171.57 A1 May 7, 2009 52 in the present specification. Depending on the location of reduction in a nocturia symptom indicates Successful treat abnormal sensory neuron activity, the toxin can be adminis ment with the composition comprising a modified Clostridial tered into e.g., the detrusor, the bladder neck including the toxin. external and internal urethral sphincters, the trigone, the blad der dome or other areas of the bladder wall, and/or other areas EXAMPLE 9 Surrounding the bladder. Such as the urethra, ureter, urogeni tal diaphragm, lower pelvic muscles, prostate, bulbourethral Treatment of Chronic Urinary Tract Infection gland, bulb, crus or penis. The patient's condition is moni 0379 A 76 year old female complains that she has urinary tored and after about 1-3 days from treatment, and the man tract infections all the time. A physician determines that the indicates that he has less difficulty in starting and/or main chronic urinary tract infections is abacterial and diagnosis the taining his ability to urinate. At one and three month check patient with urogential disorder having a neurological com ups, the man indicates that he still experiences less difficulty ponent involving abnormal sensory neuron activity. The in starting and/or maintaining his ability to urinate. This woman is treated by injecting urethroscopically a composi reduction in a urinary hesitancy symptom indicates Success tion comprising a modified Clostridial toxin as disclosed in ful treatment with the composition comprising a modified the present specification. Depending on the location of abnor Clostridial toxin. mal sensory neuron activity, the toxin can be administered into e.g., the detrusor, the bladder neck including the external EXAMPLE 7 and internal urethral sphincters, the trigone, the bladder dome or other areas of the bladder wall, and/or other areas Sur Treatment of Polyuria rounding the bladder, such as the urethra, ureter, urogenital 0377. A 68 year old male complains that he has to urinate diaphragm, or lower pelvic muscles. The patient's condition all the time during the day. A physician diagnosis the patient is monitored and after about 1-3 days from treatment, and the with polyuria having a neurological component involving physician indicates that she does not have a urinary tract abnormal sensory neuron activity. The man is treated by infection. At one and three month check-ups, the woman injecting urethroscopically a composition comprising a indicates that she has not had a urinary tract infection since modified Clostridial toxin as disclosed in the present specifi the treatment. This reduction in a urinary tract infection cation. Depending on the location of abnormal sensory neu symptom indicates Successful treatment with the composi ron activity, the toxin can be administered into e.g., the detru tion comprising a modified Clostridial toxin. Sor, the bladder neck including the external and internal 0380 A 75 year old female complains that she has urinary urethral sphincters, the trigone, the bladder dome or other tract infections all the time. A physician determines that the areas of the bladder wall, and/or other areas surrounding the chronic urinary tract infection is due to vesicoureteral reflux bladder, Such as the urethra, ureter, urogenital diaphragm, and diagnosis the patient with urogential disorder having a lower pelvic muscles, prostate, bulbourethral gland, bulb, neurological component involving abnormal sensory neuron crus or penis. The patient's condition is monitored and after activity. The woman is treated by injecting urethroscopically about 1-3 days from treatment, and the man indicates that a composition comprising a modified Clostridial toxin as does not have to urinate as many times during the day as disclosed in the present specification. Depending on the loca before the treatment. At one and three month check-ups, the tion of abnormal sensory neuron activity, the toxin can be man still indicates that does not have tourinate as many times administered into e.g., the detrusor, the bladder neck includ during the day as before the treatment. This reduction in a ing the external and internal urethral sphincters, the trigone, polyuria symptom indicates successful treatment with the the bladder dome or other areas of the bladder wall, and/or composition comprising a modified Clostridial toxin. other areas Surrounding the bladder, Such as the urethra, ure ter, urogenital diaphragm, or lower pelvic muscles. The EXAMPLE 8 patient's condition is monitored and after about 1-3 days from treatment, and the physician determines that the abnormal Treatment of Nocturia backup of urine from the bladder to the kidneys is reduced in 0378. A 57 year old female complains that she has to wake the patient. At one and three month check-ups, the woman up several times during the night in order to urinate. A phy indicates that she has not had a urinary tract infection since sician diagnosis the patient with nocturia having a neurologi the treatment. This reduction in a urinary tract infection cal component involving abnormal sensory neuron activity. symptom indicates Successful treatment with the composi The woman is treated by injecting urethroscopically a com tion comprising a modified Clostridial toxin. position comprising a modified Clostridial toxin as disclosed in the present specification. Depending on the location of EXAMPLE 10 abnormal sensory neuron activity, the toxin can be adminis Treatment of Urogenital Disorder Associated with a tered into e.g., the detrusor, the bladder neck including the Prostate Disorder external and internal urethral sphincters, the trigone, the blad der dome or other areas of the bladder wall, and/or other areas 0381. A 78 year old male complains that he has difficulty Surrounding the bladder. Such as the urethra, ureter, urogeni starting and/or maintaining his ability to urinate. A physician tal diaphragm, or lower pelvic muscles. The patient's condi determines that he has benign prostatic hyperplasia and that tion is monitored and after about 1-3 days from treatment, and this enlargement is blocking the normal flow of urine. The the woman indicates that she does not have to get up as many physician diagnosis the patient with urinary hesitancy asso times during the night to urinate as she did before the treat ciated with benign prostatic hyperplasia having a neurologi ment. At one and three month check-ups, the woman still cal component involving abnormal sensory neuron activity. indicates that she does not have to get up as many times during The man is treated by injecting a composition comprising a the night to urinate as she did before the treatment. This modified Clostridial toxin as disclosed in the present specifi US 2009/01171.57 A1 May 7, 2009 cation into the prostate and/or in the Surrounding area of the 0384. A 12 year old male diagnosed with spina bifida prostate depending on the location of abnormal sensory neu complains about the inability to control the passage of urine. ron activity. The patient's condition is monitored and after A physician determines that this urinary incontinence is due about 1-2 weeks from the treatment, the man indicates that he to his spina bifida and diagnosis the patient with urogential has less difficulty in starting and/or maintaining his ability to disorder associated with a neurogenic dysfunction having a urinate. The physician determines that the size of the prostate neurological component involving abnormal sensory neuron has reduced since the treatment. At one and three month activity. The boy is treated by injecting urethroscopically a check-ups, the man indicates that he still experiences less composition comprising a modified Clostridial toxin as dis difficulty in starting and/or maintaining his ability to urinate. closed in the present specification. Depending on the location This reduction in a urinary hesitancy symptom associated of abnormal sensory neuron activity, the toxin can be admin with benign prostatic hyperplasia indicates successful treat istered into e.g., the detrusor, the bladder neck including the ment with the composition comprising a modified Clostridial external and internal urethral sphincters, the trigone, the blad toxin. der dome or other areas of the bladder wall, and/or other areas Surrounding the bladder, Such as the urethra, ureter, urogeni EXAMPLE 11 tal diaphragm, lower pelvic muscles, prostate, bulbourethral gland, bulb, crus or penis. The patient's condition is moni Treatment of Urogenital Disorder Associated with a tored and after about 1-3 days from treatment, and the boy Neurogenic Dysfunction indicates that he has an increased ability to control the pas 0382. A 81 year old female diagnosed with Parkinson's sage or urine. At one and three month check-ups, the boy Disease complains about having a Sudden need to urinate. A indicates that he still experiences an increased ability to con physician determines that this urinary urgency is due to her trol the passage or urine. This reduction in a urogenital dis Parkinson's Disease and diagnosis the patient with urogential order symptom associated with a neurogenic dysfunction disorder associated with a neurogenic dysfunction having a indicates Successful treatment with the composition compris neurological component involving abnormal sensory neuron ing a modified Clostridial toxin. activity. The woman is treated by injecting urethroscopically 0385 A84 year old male who experienced a stroke com a composition comprising a modified Clostridial toxin as plains about not being able to urinate. A physician determines disclosed in the present specification. Depending on the loca that this urinary retention is due to his stroke and diagnosis the tion of abnormal sensory neuron activity, the toxin can be patient with urogential disorder associated with a neurogenic administered into e.g., the detrusor, the bladder neck includ dysfunction having a neurological component involving ing the external and internal urethral sphincters, the trigone, abnormal sensory neuron activity. The man is treated by the bladder dome or other areas of the bladder wall, and/or injecting urethroscopically a composition comprising a other areas Surrounding the bladder, Such as the urethra, ure modified Clostridial toxin as disclosed in the present specifi ter, urogenital diaphragm, or lower pelvic muscles. The cation. Depending on the location of abnormal sensory neu patient's condition is monitored and after about 1-3 days from ron activity, the toxin can be administered into e.g., the detru treatment, and the woman indicates that there is a reduction in Sor, the bladder neck including the external and internal the Sudden need to urinate. At one and three month check-ups, urethral sphincters, the trigone, the bladder dome or other the woman indicates that she continues to experience a areas of the bladder wall, and/or other areas surrounding the reduced Sudden need to urinate. This reduction in a urogenital bladder, such as the urethra, ureter, urogenital diaphragm, disorder symptom associated with a neurogenic dysfunction lower pelvic muscles, prostate, bulbourethral gland, bulb, indicates Successful treatment with the composition compris crus or penis. The patient's condition is monitored and after ing a modified Clostridial toxin. about 1-3 days from treatment, and the man indicates that he 0383. A 39 year old female diagnosed with multiple scle can urinate. At one and three month check-ups, the man rosis complains about having a need to urinate all the time. A indicates that he continues to experience the ability to urinate. physician determines that this urinary frequency is due to her This reduction in a urogenital disorder symptom associated multiple Sclerosis and diagnosis the patient with urogential with a neurogenic dysfunction indicates Successful treatment disorder associated with a neurogenic dysfunction having a with the composition comprising a modified Clostridial neurological component involving abnormal sensory neuron toxin. activity. The woman is treated by injecting urethroscopically 0386 A 23 year old man suffering from a spinal cord a composition comprising a modified Clostridial toxin as injury resulting from a car accident complains about the disclosed in the present specification. Depending on the loca inability to control the passage of urine. A physician deter tion of abnormal sensory neuron activity, the toxin can be mines that this urinary incontinence is due to his spinal cord administered into e.g., the detrusor, the bladder neck includ injury and diagnosis the patient with urogential disorder asso ing the external and internal urethral sphincters, the trigone, ciated with a neurogenic dysfunction having a neurological the bladder dome or other areas of the bladder wall, and/or component involving abnormal sensory neuron activity. The other areas Surrounding the bladder, Such as the urethra, ure man is treated by injecting urethroscopically a composition ter, urogenital diaphragm, or lower pelvic muscles. The comprising a modified Clostridial toxin as disclosed in the patient's condition is monitored and after about 1-3 days from present specification. Depending on the location of abnormal treatment, and the woman indicates that there is a reduction in sensory neuron activity, the toxin can be administered into the need to urinate all the time. At one and three month e.g., the detrusor, the bladder neck including the external and check-ups, the woman indicates that she still experiences a internal urethral sphincters, the trigone, the bladder dome or reduced need to urinate all the time. This reduction in a other areas of the bladder wall, and/or other areas surrounding urogenital disorder symptom associated with a neurogenic the bladder, Such as the urethra, ureter, urogenital diaphragm, dysfunction indicates successful treatment with the compo or lower pelvic muscles. The patient's condition is monitored sition comprising a modified Clostridial toxin. and after about 1-3 days from treatment, and the man indi US 2009/01171.57 A1 May 7, 2009 54 cates that he has an increased ability to control the passage or other areas of the bladder wall, and/or other areas surrounding urine. At one and three month check-ups, the man indicates the bladder, Such as the urethra, ureter, urogenital diaphragm, that he still experiences an increased ability to control the lower pelvic muscles, prostate, bulbourethral gland, bulb, passage or urine. This reduction in a urogenital disorder crus or penis. The patient's condition is monitored and after symptom associated with a neurogenic dysfunction indicates about 1-3 days from treatment, and the man indicates that Successful treatment with the composition comprising a there is a reduction in the need to urinate all the time. At one modified Clostridial toxin. and three month check-ups, the man indicates that he still 0387. A 63 year old male who has cancerous lesion in his experiences a reduced need to urinate all the time. This reduc brain complains about having a need to urinate all the time. A tion in a urogenital disorder symptom associated with a neu physician determines that this urinary frequency is due to his rogenic dysfunction indicates successful treatment with the lesion and diagnosis the patient with urogential disorder asso composition comprising a modified Clostridial toxin. ciated with a neurogenic dysfunction having a neurological 0388. The foregoing description of the invention is exem component involving abnormal sensory neuron activity. The plary for purposes of illustration and explanation. It will be man is treated by injecting urethroscopically a composition apparent to those skilled in the art that changes and modifi comprising a modified Clostridial toxin as disclosed in the cations are possible without departing from the spirit and present specification. Depending on the location of abnormal scope of the invention. All documents cited herein are hereby sensory neuron activity, the toxin can be administered into incorporated by reference. It is intended that the following e.g., the detrusor, the bladder neck including the external and claims be interpreted to embrace all Such changes and modi internal urethral sphincters, the trigone, the bladder dome or fications.
SEQUENCE LISTING
<16 Oc NUMBER OF SEO ID NOS : 125
<210 SEQ ID NO 1 <211 LENGTH: 1296 &212> TYPE: PRT <213> ORGANISM: Clostridium botulinum Serotype A &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (448) <223> OTHER INFORMATION: Light chain comprising the enzymatic domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (449) . . . (860 <223> OTHER INFORMATION: Amino-terminal half of heavy chain comprising the translocation domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (861) . . . (1296) <223> OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain.
<4 OO SEQUENCE: 1 Met Pro Phe Val Asn Lys Glin Phe Asn Tyr Lys Asp Pro Wall Asn Gly 1. 5 1O 15
Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Glin Met Glin Pro
Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 4 O 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Lieu. Asn Pro Pro Pro Glu SO 55 60
Ala Lys Glin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 7s
Asp Asn. Glu Lys Asp Asn Tyr Lieu Lys Gly Val Thir Lys Leul Phe Glu 85 90 95
Arg Ile Tyr Ser Thr Asp Lieu. Gly Arg Met Lieu. Lieu. Thir Ser Ile Wall 1OO 105 11 O
Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Lieu Lys 115 12 O 125
Val Ile Asp Thr Asn. Cys Ile Asn Val Ile Glin Pro Asp Gly Ser Tyr 13 O 135 14 O US 2009/01171.57 A1 May 7, 2009 55
- Continued Arg Ser Glu Glu Lieu. Asn Lieu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160 Ile Glin Phe Glu. Cys Lys Ser Phe Gly His Glu Val Lieu. Asn Lieu. Thr 1.65 17O 17s Arg Asn Gly Tyr Gly Ser Thr Glin Tyr Ile Arg Phe Ser Pro Asp Phe 18O 185 190 Thr Phe Gly Phe Glu Glu Ser Lieu. Glu Val Asp Thr Asn Pro Leu Lieu. 195 2 OO 2O5 Gly Ala Gly Llys Phe Ala Thr Asp Pro Ala Val Thir Lieu Ala His Glu 210 215 22O Lieu. Ile His Ala Gly His Arg Lieu. Tyr Gly Ile Ala Ile Asin Pro Asn 225 23 O 235 24 O Arg Val Phe Llys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Lieu. 245 250 255 Glu Val Ser Phe Glu Glu Lieu. Arg Thr Phe Gly Gly His Asp Ala Lys 26 O 265 27 O Phe Ile Asp Ser Lieu. Glin Glu Asn. Glu Phe Arg Lieu. Tyr Tyr Tyr Asn 27s 28O 285 Llys Phe Lys Asp Ile Ala Ser Thr Lieu. Asn Lys Ala Lys Ser Ile Val 290 295 3 OO Gly. Thir Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys 3. OS 310 315 32O Tyr Lieu. Lieu. Ser Glu Asp Thir Ser Gly Llys Phe Ser Val Asp Llys Lieu. 3.25 330 335 Llys Phe Asp Llys Lieu. Tyr Lys Met Lieu. Thr Glu Ile Tyr Thr Glu Asp 34 O 345 350 Asn Phe Val Llys Phe Phe Llys Val Lieu. Asn Arg Llys Thr Tyr Lieu. Asn 355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn. Ile Val Pro Llys Val Asn Tyr 37O 375 38O Thir Ile Tyr Asp Gly Phe Asn Lieu. Arg Asn. Thir Asn Lieu Ala Ala Asn 385 390 395 4 OO Phe Asin Gly Glin Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Lieu. 4 OS 410 415 Lys Asn. Phe Thr Gly Lieu. Phe Glu Phe Tyr Lys Lieu. Lieu. CyS Val Arg 42O 425 43 O Gly Ile Ile Thir Ser Llys Thr Lys Ser Lieu. Asp Llys Gly Tyr Asn Lys 435 4 4 O 445 Ala Lieu. Asn Asp Lieu. Cys Ile Llys Val Asn. Asn Trp Asp Lieu. Phe Phe 450 45.5 460 Ser Pro Ser Glu Asp Asn. Phe Thr Asn Asp Lieu. Asn Lys Gly Glu Glu 465 470 47s 48O Ile Thir Ser Asp Thr Asn. Ile Glu Ala Ala Glu Glu Asn. Ile Ser Lieu. 485 490 495 Asp Lieu. Ile Glin Glin Tyr Tyr Lieu. Thr Phe Asn Phe Asp Asn Glu Pro 5 OO 5 OS 510 Glu Asn. Ile Ser Ile Glu Asn Lieu. Ser Ser Asp Ile Ile Gly Glin Lieu. 515 52O 525 Glu Lieu Met Pro Asn. Ile Glu Arg Phe Pro Asn Gly Llys Llys Tyr Glu 53 O 535 54 O Lieu. Asp Llys Tyr Thr Met Phe His Tyr Lieu. Arg Ala Glin Glu Phe Glu US 2009/01171.57 A1 May 7, 2009 56
- Continued
5.45 550 555 560 His Gly Lys Ser Arg Ile Ala Lieu. Thir Asn. Ser Val Asn. Glu Ala Lieu. 565 st O sfs Lieu. Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 58O 585 590 Llys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Lieu. Gly Trp Val Glu 595 6 OO 605 Gln Leu Val Tyr Asp Phe Thr Asp Glu Thir Ser Glu Val Ser Thr Thr 610 615 62O Asp Llys Ile Ala Asp Ile Thir Ile Ile Ile Pro Tyr Ile Gly Pro Ala 625 630 635 64 O Lieu. Asn. Ile Gly Asn Met Lieu. Tyr Lys Asp Asp Phe Val Gly Ala Lieu. 645 650 655 Ile Phe Ser Gly Ala Val Ile Leu Lieu. Glu Phe Ile Pro Glu Ile Ala 660 665 670 Ile Pro Val Lieu. Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 68O 685 Val Lieu. Thr Val Glin Thir Ile Asp Asn Ala Lieu. Ser Lys Arg Asn. Glu 690 695 7 OO Llys Trp Asp Glu Val Tyr Llys Tyr Ile Val Thr Asn Trp Lieu Ala Lys 7 Os 71O 71s 72O Val Asn Thr Glin Ile Asp Lieu. Ile Arg Llys Llys Met Lys Glu Ala Lieu. 72 73 O 73 Glu Asn Glin Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Glin Tyr Asn 740 74. 75O Glin Tyr Thr Glu Glu Glu Lys Asn. Asn. Ile Asn. Phe Asn. Ile Asp Asp 75s 760 765 Lieu. Ser Ser Lys Lieu. Asn. Glu Ser Ile Asn Lys Ala Met Ile Asn. Ile 770 775 78O Asn Llys Phe Lieu. Asn Gln Cys Ser Val Ser Tyr Lieu Met Asn Ser Met 78s 79 O 79. 8OO Ile Pro Tyr Gly Wall Lys Arg Lieu. Glu Asp Phe Asp Ala Ser Lieu Lys 805 810 815 Asp Ala Lieu. Lieu Lys Tyr Ile Tyr Asp Asin Arg Gly. Thir Lieu. Ile Gly 82O 825 830 Glin Val Asp Arg Lieu Lys Asp Llys Val Asn. Asn. Thir Lieu. Ser Thr Asp 835 84 O 845 Ile Pro Phe Glin Lieu. Ser Llys Tyr Val Asp Asn Glin Arg Lieu. Lieu. Ser 850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Lieu. Asn 865 87O 87s 88O Lieu. Arg Tyr Glu Ser Asn His Lieu. Ile Asp Lieu. Ser Arg Tyr Ala Ser 885 890 895 Lys Ile Asn. Ile Gly Ser Llys Val Asn. Phe Asp Pro Ile Asp Lys Asn 9 OO 9 OS 910 Glin Ile Glin Lieu. Phe Asn Lieu. Glu Ser Ser Lys Ile Glu Val Ile Lieu. 915 92O 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 94 O Phe Trp Ile Arg Ile Pro Llys Tyr Phe Asin Ser Ile Ser Lieu. Asn Asn 945 950 955 96.O US 2009/01171.57 A1 May 7, 2009 57
- Continued
Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Llys Val 965 97O 97. Ser Lieu. Asn Tyr Gly Glu Ile Ile Trp Thr Lieu. Glin Asp Thr Glin Glu 98O 985 990 le Lys Glin Arg Val Val Phe Llys Tyr Ser Gln Met Ile Asin Ile Ser
Asp Tyr Ile Asn Arg Trp Ile Phe Val Thir Ile Thr Asn. Asn Arg Lieu
Asn Asn. Ser Lys Ile Tyr Ile Asin Gly Arg Lieu. Ile Asp Glin Llys Pro O25 O3 O O35 104 O le Ser Asn Lieu. Gly Asn. Ile His Ala Ser Asn. Asn. Ile Met Phe Lys
Lieu. Asp Gly Cys Arg Asp Thr His Arg Tyr e Trp Ile Llys Tyr Phe
Asn Lieu. Phe Asp Llys Glu Lieu. Asn. Glu Lys Glu Ile Lys Asp Lieu. Tyr
Asp Asn Glin Ser Asn. Ser Gly Ile Lieu Lys Asp Phe Trp Gly Asp Tyr
Lieu. Glin Tyr Asp Llys Pro Tyr Tyr Met Lieu. Asn Lieu. Tyr Asp Pro Asn OS 10 15 112O Llys Tyr Val Asp Wall Asn. Asn Val Gly Ile Arg Gly Tyr Met Tyr Lieu.
Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Lieu. Asn Ser
Ser Lieu. Tyr Arg Gly hir Llys Phe Ile Ile Llys Llys Tyr Ala Ser Gly
Asn Lys Asp Asn. Ile Val Arg Asn. Asn Asp Arg Val Tyr Ile Asin Val
Val Val Lys Asn Lys Glu Tyr Arg Lieu Ala Thr Asn Ala Ser Glin Ala 85 9 O 95 12 OO Gly Val Glu Lys Ile Lieu. Ser Ala Lieu. Glu e Pro Asp Val Gly Asn
Lieu. Ser Glin Val Val Val Met Lys Ser Lys Asn Asp Glin Gly Ile Thr
Asn Lys Cys Llys Met Asn Lieu. Glin Asp Asn. Asn Gly Asn Asp Ile Gly
Phe Ile Gly Phe His Glin Phe Asin Asn. Ile Ala Lys Lieu Val Ala Ser
Asn Trp Tyr Asn Arg Glin Ile Glu Arg Ser Ser Arg Thr Lieu. Gly Cys 265 27 O 27s 128O Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Lieu.
<210 SEQ ID NO 2 <211 LENGTH: 1291 &212> TYPE: PRT <213> ORGANISM: Clostridium botulinum Serotype B &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) ... (441) <223> OTHER INFORMATION: Light chain comprising the enzymatic domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (442) ... (847) <223> OTHER INFORMATION: Amino-terminal half of heavy chain comprising US 2009/01171.57 A1 May 7, 2009 58
- Continued
the translocation domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (848) . . . (1291) <223> OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain.
<4 OO SEQUENCE: 2 Met Pro Val Thir Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1. 5 1O 15 Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 2O 25 3 O Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 4 O 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly SO 55 6 O le Phe Asin Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Lieu. Asn 65 70 7s 8O Thir Asn Asp Llys Lys Asn. Ile Phe Lieu. Glin Thr Met Ile Llys Lieu. Phe 85 9 O 95 Asn Arg Ile Llys Ser Llys Pro Lieu. Gly Glu Lys Lieu. Lieu. Glu Met Ile OO OS 1O le Asin Gly Ile Pro Tyr Lieu. Gly Asp Arg Arg Val Pro Lieu. Glu Glu 15 2O 25 Phe ASn Thr ASn Ile Ala Ser Val Thr Val ASn Llys Lieu. Ile Ser Asn 3O 35 4 O Pro Gly Glu Val Glu Arg Llys Lys Gly Ile Phe Ala Asn Lieu. Ile Ile 45 SO 55 160 Phe Gly Pro Gly Pro Val Lieu. Asn Glu Asin Glu Thir Ile Asp Ile Gly 65 70 7s le Glin Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Glin 8O 85 90 Met Llys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Glin Glu 95 2 OO 2O5 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 22O Ala Lieu. Ile Lieu Met His Glu Lieu. Ile His Val Lieu. His Gly Lieu. Tyr 225 23 O 235 24 O Gly Ile Llys Val Asp Asp Lieu Pro Ile Val Pro Asn. Glu Lys Llys Phe 245 250 255 Phe Met Glin Ser Thr Asp Ala Ile Glin Ala Glu Glu Lieu. Tyr Thr Phe 26 O 265 27 O Gly Gly Glin Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Llys Ser Ile 27s 28O 285 Tyr Asp Llys Val Lieu. Glin Asn. Phe Arg Gly Ile Val Asp Arg Lieu. Asn 290 295 3 OO Llys Val Lieu Val Cys Ile Ser Asp Pro Asn. Ile Asn. Ile Asn. Ile Tyr 3. OS 310 315 32O Lys Asn Llys Phe Lys Asp Llys Tyr Llys Phe Val Glu Asp Ser Glu Gly 3.25 330 335 Llys Tyr Ser Ile Asp Val Glu Ser Phe Asp Llys Lieu. Tyr Lys Ser Lieu. 34 O 345 350 Met Phe Gly Phe Thr Glu Thir Asn Ile Ala Glu Asn Tyr Lys Ile Llys 355 360 365 US 2009/01171.57 A1 May 7, 2009 59
- Continued
Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Llys 37O 375 38O Asn Lieu. Lieu. Asp Asn. Glu Ile Tyr Thir Ile Glu Glu Gly Phe Asn. Ile 385 390 395 4 OO Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Glin Asn Lys Ala Ile 4 OS 410 415 Asn Lys Glin Ala Tyr Glu Glu Ile Ser Lys Glu. His Lieu Ala Val Tyr 42O 425 43 O Lys Ile Gln Met Cys Llys Ser Wall Lys Ala Pro Gly Ile Cys Ile Asp 435 4 4 O 445 Val Asp Asn. Glu Asp Lieu. Phe Phe Ile Ala Asp Lys Asn. Ser Phe Ser 450 45.5 460 Asp Asp Lieu. Ser Lys Asn. Glu Arg Ile Glu Tyr Asn Thr Glin Ser Asn 465 470 47s 48O Tyr Ile Glu Asn Asp Phe Pro Ile Asn. Glu Lieu. Ile Lieu. Asp Thr Asp 485 490 495 Lieu. Ile Ser Lys Ile Glu Lieu Pro Ser Glu Asn Thr Glu Ser Lieu. Thr 5 OO 5 OS 510 Asp Phe Asin Val Asp Val Pro Val Tyr Glu Lys Glin Pro Ala Ile Llys 515 52O 525 Lys Ile Phe Thr Asp Glu Asn. Thir Ile Phe Glin Tyr Lieu. Tyr Ser Glin 53 O 535 54 O Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Lieu. Thir Ser Ser Phe Asp 5.45 550 555 560 Asp Ala Lieu. Lieu. Phe Ser Asn Llys Val Tyr Ser Phe Phe Ser Met Asp 565 st O sfs Tyr Ile Llys Thir Ala Asn Llys Val Val Glu Ala Gly Lieu. Phe Ala Gly 58O 585 590 Trp Val Lys Glin Ile Val Asn Asp Phe Val Ile Glu Ala Asn Llys Ser 595 6 OO 605 Asn Thr Met Asp Llys Ile Ala Asp Ile Ser Lieu. Ile Val Pro Tyr Ile 610 615 62O Gly Lieu Ala Lieu. Asn Val Gly Asn. Glu Thir Ala Lys Gly Asn. Phe Glu 625 630 635 64 O Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Lieu. Lieu. Glu Phe Ile Pro 645 650 655 Glu Lieu. Lieu. Ile Pro Val Val Gly Ala Phe Lieu. Lieu. Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Llys Thir Ile Asp Asn Ala Lieu. Thir Lys 675 68O 685 Arg Asn. Glu Lys Trp Ser Asp Met Tyr Gly Lieu. Ile Val Ala Glin Trp 690 695 7 OO Lieu. Ser Thr Val Asn Thr Glin Phe Tyr Thr Ile Lys Glu Gly Met Tyr 7 Os 71O 71s 72O Lys Ala Lieu. Asn Tyr Glin Ala Glin Ala Lieu. Glu Glu Ile Ile Llys Tyr 72 73 O 73 Arg Tyr Asn. Ile Tyr Ser Glu Lys Glu Lys Ser Asn. Ile Asn. Ile Asp 740 74. 75O Phe Asn Asp Ile Asn. Ser Llys Lieu. Asn. Glu Gly Ile Asn Glin Ala Ile 75s 760 765 US 2009/01171.57 A1 May 7, 2009 60
- Continued Asp Asn. Ile Asn. Asn. Phe Ile Asin Gly Cys Ser Val Ser Tyr Lieu Met 770 775 78O Llys Llys Met Ile Pro Lieu Ala Val Glu Lys Lieu. Lieu. Asp Phe Asp Asn 78s 79 O 79. 8OO Thir Lieu Lys Lys Asn Lieu. Lieu. Asn Tyr Ile Asp Glu Asn Llys Lieu. Tyr 805 810 815 Lieu. Ile Gly Ser Ala Glu Tyr Glu Lys Ser Llys Val Asn Llys Tyr Lieu. 82O 825 830 Lys. Thir Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thir Ile 835 84 O 845 Lieu. Ile Glu Met Phe Asn Llys Tyr Asn. Ser Glu Ile Lieu. Asn. Asn. Ile 850 855 860 Ile Lieu. Asn Lieu. Arg Tyr Lys Asp Asn. Asn Lieu. Ile Asp Lieu. Ser Gly 865 87O 87s 88O Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Lieu. Asn Asp Llys 885 890 895 Asn Glin Phe Llys Lieu. Thir Ser Ser Ala Asn. Ser Lys Ile Arg Val Thr 9 OO 9 OS 910 Gln Asn Glin Asn Ile Ile Phe Asin Ser Val Phe Lieu. Asp Phe Ser Val 915 92O 925 Ser Phe Trp Ile Arg Ile Pro Llys Tyr Lys Asn Asp Gly Ile Glin Asn 930 935 94 O Tyr Ile His Asn Glu Tyr Thr Ile Ile Asin Cys Met Lys Asn Asn Ser 945 950 955 96.O Gly Trp Llys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thir Lieu. Ile 965 97O 97. Asp Ile Asin Gly Llys Thr Lys Ser Val Phe Phe Glu Tyr Asn. Ile Arg 98O 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thir Ile Thr
Asn Asn Lieu. Asn. Asn Ala Lys Ile Tyr Ile Asn Gly Llys Lieu. Glu Ser
Asn. Thir Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile O25 O3 O O35 104 O le Phe Llys Lieu. Asp Gly Asp Ile Asp Arg Thr Glin Phe Ile Trp Met O45 OSO O55 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Lieu Ser Glin Ser Asn Ile Glu O60 O65 Of O Glu Arg Tyr Lys Ile Glin Ser Tyr Ser Glu Tyr Lieu Lys Asp Phe Trp O7s O8O O85 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly O90 O95 1OO Asn Lys Asn. Ser Tyr Ile Llys Lieu Lys Lys Asp Ser Pro Val Gly Glu OS 11 O 115 112O e Lieu. Thir Arg Ser Llys Tyr Asn Glin Asn. Ser Llys Tyr Ile Asn Tyr 25 13 O 135 Arg Asp Lieu. Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Llys Ser Asn 4 O 145 15 O Ser Glin Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr 55 16 O 1.65 Lieu. Asp Phe Phe Asn Lieu. Asn Glin Glu Trp Arg Val Tyr Thr Tyr Lys US 2009/01171.57 A1 May 7, 2009 61
- Continued
18O
Glu Luell Phe Lel Ala Pro Ile Ser Asp 195 12 OO
Asp Glu Phe Tyr Ile Glin Ile Lys Glu Asp Glu Glin 215
Ser Cys Luell Phe Lys Lys Asp Glu Glu Ser Thr 23 O
Glu Ile Gly Lell Ile His Arg Phe Tyr Glu Ser Gly Ile 245
Phe Glu Glu Tyr Phe Cys le Ser Trp Tyr Lieu. 26 O
Glu Wall Asn Luell Lys Lell Gly Asn Trp 27s 128O
Phe Ile Pro Lys Gly Trp Thir Glu
> SEQ ID NO 3 > LENGTH: 1291 > TYPE : PRT > ORGANISM: Clostridium botulinum Serotype C1 > FEATURE; > NAME/KEY: DOMAIN > LOCATION: (1) ... (449) > OTHER INFORMATION: Light chain comprising the enzymatic domain. > NAME/KEY: DOMAIN > LOCATION: (45O) . . . (855) > OTHER INFORMATION: Amino-terminal half of heavy chain comprising the translocation domain. > NAME/KEY: DOMAIN > LOCATION: (856) . . . (1291) > OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain.
<4 OO SEQUENCE: 3
Met Pro Ile Thir Ile Asn Asn Phe Asn Tyr Ser Asp Pro Wall Asp Asn 1. 5 15
Lys ASn Ile Luell Tyr Lell Asp Thir His Luell ASn Thir Lell Ala Asn. Glu 2O 25 3 O
Pro Glu Lys Ala Phe Arg Ile Thir Gly Asn Ile Trp Wall Ile Pro Asp 35 4 O 45
Arg Phe Ser Arg Asn Ser Asn Pro Asn Luell ASn Pro Pro Arg Val SO 55 6 O
Thir Ser Pro Ser Gly Asp Pro ASn Lell Ser Thir Asp 65 70 7s
Ser Asp Llys Asp Pro Phe Lell Glu Ile le Lell Phe 85 9 O
Ile Asn. Ser Arg Glu Ile Gly Glu Glu Luell le Arg Luell Ser Thr 1 OO 105
Asp Ile Pro Phe Pro Gly Asn Asn Asn Thir Pro Ile Asn Thir Phe Asp 115 12O 25
Phe Asp Wall Asp Phe Asn Ser Wall Asp Wall Lys Thir Arg Glin Gly Asn 13 O 135 4 O
Asn Trp Val Lys Thir Gly Ser Ile Asn Pro Ser Wall Ile Ile Thr Gly 145 150 55 160
Pro Arg Glu Asn Ile Ile Asp Pro Glu Thir Ser Thir Phe Lieu. Thir 1.65 17O 7s US 2009/01171.57 A1 May 7, 2009 62
- Continued Asn Asn. Thir Phe Ala Ala Glin Glu Gly Phe Gly Ala Lieu. Ser Ile Ile 18O 185 190 Ser Ile Ser Pro Arg Phe Met Lieu. Thr Tyr Ser Asn Ala Thr Asn Asp 195 2 OO 2O5 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 22O Lieu. Ile Lieu Met His Glu Lieu. Asn His Ala Met His Asn Lieu. Tyr Gly 225 23 O 235 24 O Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 Phe Tyr Ser Glin Tyr Asn Val Lys Lieu. Glu Tyr Ala Glu Ile Tyr Ala 26 O 265 27 O Phe Gly Gly Pro Thir Ile Asp Lieu. Ile Pro Llys Ser Ala Arg Llys Tyr 27s 28O 285 Phe Glu Glu Lys Ala Lieu. Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Lieu. 290 295 3 OO Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly 3. OS 310 315 32O Glu Tyr Lys Glin Llys Lieu. Ile Arg Llys Tyr Arg Phe Val Val Glu Ser 3.25 330 335 Ser Gly Glu Val Thr Val Asn Arg Asn Llys Phe Val Glu Lieu. Tyr Asn 34 O 345 350 Glu Lieu. Thr Glin Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Glin Asn Arg Lys Ile Tyr Lieu Ser Asn Val Tyr Thr Pro Val Thr 37O 375 38O Ala Asn. Ile Lieu. Asp Asp Asn Val Tyr Asp Ile Glin Asn Gly Phe Asn 385 390 395 4 OO Ile Pro Llys Ser Asn Lieu. Asn Val Lieu. Phe Met Gly Glin Asn Lieu. Ser 4 OS 410 415 Arg ASn Pro Ala Lieu. Arg Llys Val Asn Pro Glu Asn Met Lieu. Tyr Lieu 42O 425 43 O Phe Thr Llys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Lieu. Tyr Asn 435 4 4 O 445 Llys Thr Lieu. Asp Cys Arg Glu Lieu. Lieu Val Lys Asn. Thir Asp Lieu Pro 450 45.5 460 Phe Ile Gly Asp Ile Ser Asp Wall Lys Thr Asp Ile Phe Lieu. Arg Llys 465 470 47s 48O Asp Ile Asin Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495 Val Asp Glin Val Ile Lieu. Ser Lys Asn. Thir Ser Glu. His Gly Glin Lieu 5 OO 5 OS 510 Asp Lieu. Lieu. Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Lieu Pro Gly 515 52O 525 Glu Asn Glin Val Phe Tyr Asp Asn Arg Thr Glin Asn. Wall Asp Tyr Lieu. 53 O 535 54 O Asn Ser Tyr Tyr Tyr Lieu. Glu Ser Glin Llys Lieu. Ser Asp Asn Val Glu 5.45 550 555 560 Asp Phe Thir Phe Thr Arg Ser Ile Glu Glu Ala Lieu. Asp Asn. Ser Ala 565 st O sfs Llys Val Tyr Thr Tyr Phe Pro Thr Lieu Ala Asn Llys Val Asn Ala Gly US 2009/01171.57 A1 May 7, 2009 63
- Continued
58O 585 590 Val Glin Gly Gly Lieu. Phe Lieu Met Trp Ala Asn Asp Val Val Glu Asp 595 6 OO 605 Phe Thir Thr Asn. Ile Lieu. Arg Lys Asp Thir Lieu. Asp Llys Ile Ser Asp 610 615 62O Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Lieu. Asn Ile Ser Asn 625 630 635 64 O Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655 Thir Ile Leu Lieu. Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670 Ala Phe Val Ile Tyr Ser Llys Val Glin Glu Arg Asn. Glu Ile Ile Llys 675 68O 685 Thir Ile Asp Asn. Cys Lieu. Glu Glin Arg Ile Lys Arg Trp Lys Asp Ser 690 695 7 OO Tyr Glu Trp Met Met Gly. Thir Trp Leu Ser Arg Ile Ile Thr Glin Phe 7 Os 71O 71s 72O Asn Asn. Ile Ser Tyr Gln Met Tyr Asp Ser Lieu. Asn Tyr Glin Ala Gly 72 73 O 73 Ala Ile Lys Ala Lys Ile Asp Lieu. Glu Tyr Lys Llys Tyr Ser Gly Ser 740 74. 75O Asp Lys Glu ASn Ile Llys Ser Glin Val Glu ASn Lieu Lys Asn Ser Lieu. 75s 760 765 Asp Wall Lys Ile Ser Glu Ala Met Asn. Asn. Ile Asn Llys Phe Ile Arg 770 775 78O Glu Cys Ser Val Thr Tyr Lieu Phe Lys Asn Met Leu Pro Llys Val Ile 78s 79 O 79. 8OO Asp Glu Lieu. Asn. Glu Phe Asp Arg Asn. Thir Lys Ala Lys Lieu. Ile Asn 805 810 815 Lieu. Ile Asp Ser His Asn. Ile Ile Lieu Val Gly Glu Val Asp Llys Lieu. 82O 825 830 Lys Ala Lys Val Asn. Asn. Ser Phe Glin Asn. Thir Ile Pro Phe Asn. Ile 835 84 O 845 Phe Ser Tyr Thir Asn. Asn. Ser Lieu. Lieu Lys Asp Ile Ile Asin Glu Tyr 850 855 860 Phe Asn. Asn. Ile Asn Asp Ser Lys Ile Lieu. Ser Lieu. Glin Asn Arg Llys 865 87O 87s 88O Asn. Thir Lieu Val Asp Thir Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895 Gly Asp Val Glin Lieu. Asn Pro Ile Phe Pro Phe Asp Phe Llys Lieu. Gly 9 OO 9 OS 910 Ser Ser Gly Glu Asp Arg Gly Llys Val Ile Val Thr Glin Asn. Glu Asn 915 92O 925 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 94 O Arg Ile Asn Llys Trp Val Ser Asn Lieu Pro Gly Tyr Thir Ile Ile Asp 945 950 955 96.O Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 97O 97. Lieu Val Phe Thir Lieu Lys Glin Asn. Glu Asp Ser Glu Glin Ser Ile Asn 98O 985 990 US 2009/01171.57 A1 May 7, 2009 64
- Continued
Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Llys Trp Phe 995 OOO OOS Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile
Asn Gly Lys Lieu. Ile Asp Thir Ile Llys Val Lys Glu Lieu. Thr Gly Ile O25 O3 O O35 104 O Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile Pro Asp Thr
Gly Lieu. Ile Thir Ser Asp Ser Asp Asn. Ile Asn Met Trp Ile Arg Asp
Phe Tyr Ile Phe Ala Lys Glu Lieu. Asp Gly Lys Asp Ile Asn. Ile Lieu.
Phe Asin Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn
Asp Lieu. Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn. Ile Asp Tyr Lieu OS 10 15 112O Asn Arg Tyr Met Tyr Ala Asn. Ser Arg Glin e Val Phe Asn Thr Arg
Arg Asn. Asn. Asn Asp Phe Asn. Glu Gly Tyr Lys Ile Ile Ile Lys Arg
e Arg Gly Asn. Thir Asn Asp Thr Arg Val Arg Gly Gly Asp Ile Lieu
yr Phe Asp Met Thr e Asn. Asn Lys Ala Tyr Asn Lieu. Phe Met Lys
Asn Glu Thir Met Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala 85 9 O 95 12 OO e Gly Lieu. Arg Glu Glin Thr Lys Asp Ile Asn Asp Asn. Ile Ile Phe
Glin Ile Glin Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Glin Ile Phe
Llys Ser Asn. Phe Asn Gly Glu Asn. Ile Ser Gly Ile Cys Ser Ile Gly
Chir Tyr Arg Phe Arg Lieu. Gly Gly Asp Trip Tyr Arg His Asn Tyr Lieu.
Val Pro Thr Val Lys Glin Gly Asn Tyr Ala Ser Lieu Lleu. Glu Ser Thr 265 27 O 27s 128O Ser Thr His Trp Gly Phe Val Pro Val Ser Glu
<210 SEQ ID NO 4 <211 LENGTH: 1276 &212> TYPE: PRT <213> ORGANISM: Clostridium botulinum Serotype D &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) ... (44.2) <223> OTHER INFORMATION: Light chain comprising the enzymatic domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (443) . . . (851) <223> OTHER INFORMATION: Amino-terminal half of heavy chain comprising the translocation domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (852) . . . (1276) <223> OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain. US 2009/01171.57 A1 May 7, 2009 65
- Continued <4 OO SEQUENCE: 4 Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp 1. 5 1O 15 Asn Asp Ile Lieu. Tyr Lieu. Arg Ile Pro Glin Asn Llys Lieu. Ile Thir Thr 2O 25 3 O Pro Val Lys Ala Phe Met Ile Thr Glin Asn Ile Trp Val Ile Pro Glu 35 4 O 45 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro SO 55 6 O Thir Ser Lys Tyr Glin Ser Tyr Tyr Asp Pro Ser Tyr Lieu Ser Thr Asp 65 70 7s 8O Glu Gln Lys Asp Thr Phe Lieu Lys Gly Ile Ile Llys Lieu. Phe Lys Arg 85 9 O 95 le Asn. Glu Arg Asp Ile Gly Lys Llys Lieu. Ile Asn Tyr Lieu Val Val OO OS 1O Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 15 2O 25 Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 3O 35 4 O Ser Trp Llys Val Thr Asn Ile Ile Thr Pro Ser Val Lieu. Ile Phe Gly 45 SO 55 160 Pro Lieu. Pro ASn Ile Lieu. Asp Tyr Thr Ala Ser Lieu. Thr Lieu. Glin Gly 65 70 7s Gln Glin Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Lieu Ser Ile Leu 8O 85 90 Llys Val Ala Pro Glu Phe Leu Lieu. Thr Phe Ser Asp Val Thr Ser Asn 95 2 OO 2O5 Gln Ser Ser Ala Val Lieu. Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 22O Ile Ala Leu Met His Glu Lieu. Thr His Ser Lieu. His Gln Leu Tyr Gly 225 23 O 235 24 O Ile Asn. Ile Pro Ser Asp Lys Arg Ile Arg Pro Glin Val Ser Glu Gly 245 250 255 Phe Phe Ser Glin Asp Gly Pro Asn Val Glin Phe Glu Glu Lieu. Tyr Thr 26 O 265 27 O Phe Gly Gly Lieu. Asp Val Glu Ile Ile Pro Glin Ile Glu Arg Ser Glin 27s 28O 285 Lieu. Arg Glu Lys Ala Lieu. Gly His Tyr Lys Asp Ile Ala Lys Arg Lieu. 290 295 3 OO Asn Asn. Ile Asn Llys Thir Ile Pro Ser Ser Trp Ile Ser Asn. Ile Asp 3. OS 310 315 32O Llys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn. Phe Asp Lys Asp Asn 3.25 330 335 Thr Gly Asn Phe Val Val Asn Ile Asp Llys Phe Asn Ser Lieu. Tyr Ser 34 O 345 350 Asp Lieu. Thir Asn Val Met Ser Glu Val Val Tyr Ser Ser Glin Tyr Asn 355 360 365 Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Lieu Pro Val Phe 37O 375 38O Ala Asn. Ile Lieu. Asp Asp Asn. Ile Tyr Thir Ile Arg Asp Gly Phe Asn 385 390 395 4 OO US 2009/01171.57 A1 May 7, 2009 66
- Continued
Lieu. Thir Asn Lys Gly Phe Asn. Ile Glu Asn. Ser Gly Glin Asn. Ile Glu 4 OS 410 415 Arg ASn Pro Ala Lieu. Glin Llys Lieu. Ser Ser Glu Ser Val Val Asp Lieu 42O 425 43 O Phe Thr Llys Val Cys Lieu. Arg Lieu. Thir Lys Asn. Ser Arg Asp Asp Ser 435 4 4 O 445 Thir Cys Ile Llys Wall Lys Asn. Asn Arg Lieu Pro Tyr Val Ala Asp Llys 450 45.5 460 Asp Ser Ile Ser Glin Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu 465 470 47s 48O Thir Asn Val Glin Asn Tyr Ser Asp Llys Phe Ser Lieu. Asp Glu Ser Ile 485 490 495 Lieu. Asp Gly Glin Val Pro Ile Asin Pro Glu Ile Val Asp Pro Lieu. Lieu. 5 OO 5 OS 510 Pro Asn Val Asn Met Glu Pro Leu. Asn Lieu Pro Gly Glu Glu Ile Val 515 52O 525 Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Lieu. Asn Ser Tyr Tyr 53 O 535 54 O Tyr Lieu. Glu Ser Glin Llys Lieu. Ser Asn. Asn Val Glu Asn. Ile Thr Lieu. 5.45 550 555 560 Thir Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 st O sfs Phe Lieu Pro Ser Lieu Ala Glu Lys Val Asn Lys Gly Val Glin Ala Gly 58O 585 590 Lieu. Phe Lieu. Asn Trp Ala Asn. Glu Val Val Glu Asp Phe Thir Thr Asn 595 6 OO 605 Ile Met Lys Lys Asp Thir Lieu. Asp Llys Ile Ser Asp Val Ser Val Ile 610 615 62O Ile Pro Tyr Ile Gly Pro Ala Lieu. Asn. Ile Gly Asn. Ser Ala Lieu. Arg 625 630 635 64 O Gly Asn. Phe Asin Glin Ala Phe Ala Thr Ala Gly Val Ala Phe Lieu. Lieu. 645 650 655 Glu Gly Phe Pro Glu Phe Thir Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670 Tyr Ser Ser Ile Glin Glu Arg Glu Lys Ile Ile Llys Thir Ile Glu Asn 675 68O 685 Cys Lieu. Glu Glin Arg Val Lys Arg Trp Lys Asp Ser Tyr Glin Trp Met 690 695 7 OO Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Glin Phe Asn His Ile Asn 7 Os 71O 71s 72O Tyr Glin Met Tyr Asp Ser Lieu. Ser Tyr Glin Ala Asp Ala Ile Lys Ala 72 73 O 73 Lys Ile Asp Lieu. Glu Tyr Llys Llys Tyr Ser Gly Ser Asp Llys Glu Asn 740 74. 75O Ile Llys Ser Glin Val Glu Asn Lieu Lys Asn. Ser Lieu. Asp Wall Lys Ile 75s 760 765 Ser Glu Ala Met Asn. Asn. Ile Asn Llys Phe Ile Arg Glu. Cys Ser Val 770 775 78O Thir Tyr Lieu. Phe Lys Asn Met Lieu Pro Llys Val Ile Asp Glu Lieu. Asn 78s 79 O 79. 8OO US 2009/01171.57 A1 May 7, 2009 67
- Continued Llys Phe Asp Lieu. Arg Thr Llys Thr Glu Lieu. Ile Asn Lieu. Ile Asp Ser 805 810 815 His Asn. Ile Ile Lieu Val Gly Glu Val Asp Arg Lieu Lys Ala Lys Val 82O 825 830 Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 84 O 845 Asn Asn. Ser Lieu Lleu Lys Asp Ile Ile Asin Glu Tyr Phe Asn. Ser Ile 850 855 860 Asn Asp Ser Lys Ile Lieu. Ser Lieu. Glin Asn Llys Lys Asn Ala Lieu Val 865 87O 87s 88O Asp Thir Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Glin 885 890 895 Lieu. Asn. Thir Ile Tyr Thr Asn Asp Phe Llys Lieu. Ser Ser Ser Gly Asp 9 OO 9 OS 910 Lys Ile Ile Val Asn Lieu. Asn. Asn. Asn. Ile Lieu. Tyr Ser Ala Ile Tyr 915 92O 925 Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Lieu. Thr 930 935 94 O Asn Ser His Asn Glu Tyr Thr Ile Ile Asin Ser Ile Glu Glin Asn Ser 945 950 955 96.O Gly Trp Llys Lieu. Cys Ile Arg Asn Gly Asn. Ile Glu Trp Ile Lieu. Glin 965 97O 97. Asp Wall Asn Arg Llys Tyr Lys Ser Lieu. Ile Phe Asp Tyr Ser Glu Ser 98O 985 990 Lieu. Ser His Thr Gly Tyr Thr Asn Llys Trp Phe Phe Val Thir Ile Thr
Asn Asn. Ile Met Gly Tyr Met Lys Lieu. Tyr Ile Asn Gly Glu Lieu Lys
Glin Ser Glin Lys Ile Glu Asp Lieu. Asp Glu Val Llys Lieu. Asp Llys Thr O25 O3 O O35 104 O le Val Phe Gly Ile Asp Glu Asn. Ile Asp Glu Asn Gln Met Lieu. Trip
le Arg Asp Phe Asn. Ile Phe Ser Lys Glu Lieu. Ser Asn. Glu Asp Ile
Asn. Ile Val Tyr Glu Gly Glin Ile Lieu. Arg Asn Val Ile Lys Asp Tyr
rp Gly Asn Pro Lieu Lys Phe Asp Thr Glu Tyr Tyr Ile Ile Asin Asp
Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Glu Ser Asn Val Lieu Val Lieu OS 10 15 112O Val Glin Tyr Pro Asp Arg Ser Lys Lieu. Tyr Thr Gly Asn Pro Ile Thr
e Llys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg Ile Lieu. Asn Gly
Asp Asn. Ile Ile Lieu. His Met Lieu. Tyr Asn. Ser Arg Llys Tyr Met Ile
e Arg Asp Thr Asp Thr Ile Tyr Ala Thr Glin Gly Gly Glu. Cys Ser
Glin Asn. Cys Val Tyr Ala Lieu Lys Lieu. Glin Ser Asn Lieu. Gly Asn Tyr 85 9 O 95 12 OO Gly Ile Gly Ile Phe Ser Ile Lys Asn. Ile Val Ser Lys Asn Llys Tyr US 2009/01171.57 A1 May 7, 2009 68
- Continued
12O5 121 O 1215
Cys Ser Glin Ile Phe Ser Ser Phe Arg Glu Asn. Thir Met Lieu. Lieu Ala 122O 1225 123 O
Asp Ile Tyr Llys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 124 O 1245
Wall Ala Wall Thir Asn Tyr Glu Thir Lys Luell Luell Ser Thir Ser Ser Phe 1250 1255 126 O
Trp Llys Phe Ile Ser Arg Asp Pro Gly Trp Wall Glu 1265 127 O 1275
<210 SEQ ID NO 5 <211 LENGTH: 1252 &212> TYPE : PRT <213> ORGANISM: Clostridium botulinum Serotype E &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . ... (422) <223> OTHER INFORMATION: Light chain comprising the enzymatic domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (423) . . . (834) <223> OTHER INFORMATION: Amino-terminal half of heavy chain comprising the translocation domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (835) . . . (1252) <223> OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain.
<4 Oos SEQUENCE: 5
Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Wall Asn Asp Arg 1. 5 15
Thir Ile Luell Tyr Ile Llys Pro Gly Gly Glin Glu Phe Ser 2O 25 3 O
Phe Asn Ile Met Lys ASn Ile Trp Ile Ile Pro Glu Arg Asn Wall Ile 35 4 O 45
Gly Thr Thir Pro Glin Asp Phe His Pro Pro Thir Ser Lell Asn Gly SO 55 6 O
Asp Ser Ser Asp Pro Asn Tyr Luell Glin Ser Asp Glu Glu Lys 65 70 7s
Asp Arg Phe Lieu Lys le Wall Thir Ile Phe Asn Arg Ile Asn Asn 85
ASn Luell Ser Gly Gly le Lieu. Luell Glu Glu Cell Ser Ala Asn Pro OO OS 1O
yr Lieu. Gly Asn Asp Asn. Thir Pro Asp Asn Glin Phe His Ile Gly Asp 15 2O 25
Ala Ser Ala Wall Glu le Llys Phe Ser Asn Gly Ser Glin Asp Ile Luell 3O 35 4 O
Leul Pro Asn Wall Ile le Met Gly Ala Glu Pro Asp Lell Phe Glu Thir 45 SO 55 160
Asn. Ser Ser ASn Ile Ser Luell Arg Asn Asn yr Met Pro Ser Asn His 70 7s
Gly Phe Gly Ser Ile Ala Ile Wall Thir Phe Ser Pro Glu Ser Phe 85 90
Arg Phe Asn Asp Asn Ser Met Asn Glu Phe le Glin Asp Pro Ala Luell 2 OO 2O5
Thir Lieu. Met His Glu. Lieu. Ile His Ser Luell His Gly Lell Gly Ala 210 215 22O US 2009/01171.57 A1 May 7, 2009 69
- Continued Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Glin Asn Pro Leu 225 23 O 235 24 O Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Lieu. Thir Phe Gly 245 250 255 Gly. Thir Asp Lieu. Asn. Ile Ile Thir Ser Ala Glin Ser Asn Asp Ile Tyr 26 O 265 27 O Thir Asn Lieu. Lieu Ala Asp Tyr Lys Lys Ile Ala Ser Llys Lieu. Ser Lys 27s 28O 285 Val Glin Val Ser ASn Pro Lieu. Lieu. Asn Pro Tyr Lys Asp Val Phe Glu 290 295 3 OO Ala Lys Tyr Gly Lieu. Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 3. OS 310 315 32O Ile Asn Llys Phe Asn Asp Ile Phe Llys Llys Lieu. Tyr Ser Phe Thr Glu 3.25 330 335 Phe Asp Leu Ala Thr Lys Phe Glin Val Lys Cys Arg Glin Thr Tyr Ile 34 O 345 350 Gly Glin Tyr Lys Tyr Phe Llys Lieu. Ser Asn Lieu. Lieu. Asn Asp Ser Ile 355 360 365 Tyr Asn. Ile Ser Glu Gly Tyr Asn. Ile Asn. Asn Lieu Lys Val Asn. Phe 37O 375 38O Arg Gly Glin Asn Ala Asn Lieu. Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 4 OO Gly Arg Gly Lieu Val Llys Lys Ile Ile Arg Phe Cys Lys Asn. Ile Val 4 OS 410 415 Ser Val Lys Gly Ile Arg Llys Ser Ile Cys Ile Glu Ile Asin Asn Gly 42O 425 43 O Glu Lieu. Phe Phe Val Ala Ser Glu Asn. Ser Tyr Asn Asp Asp Asn. Ile 435 4 4 O 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thir Ser Asn Asn Asn Tyr 450 45.5 460 Glu Asn Asp Lieu. Asp Glin Val Ile Lieu. Asn. Phe Asn. Ser Glu Ser Ala 465 470 47s 48O Pro Gly Lieu. Ser Asp Glu Lys Lieu. Asn Lieu. Thir Ile Glin Asn Asp Ala 485 490 495 Tyr Ile Pro Llys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 5 OO 5 OS 510 Asp Wall Asn. Glu Lieu. Asn Val Phe Phe Tyr Lieu. Asp Ala Glin Llys Val 515 52O 525 Pro Glu Gly Glu Asn. Asn. Wall Asn Lieu. Thir Ser Ser Ile Asp Thir Ala 53 O 535 54 O Lieu. Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 5.45 550 555 560 Asn Asn Val Asn Llys Pro Val Glin Ala Ala Lieu. Phe Val Ser Trp Ile 565 st O sfs Gln Glin Val Lieu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 58O 585 590 Val Asp Llys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Lieu 595 6 OO 605 Ala Lieu. Asn. Ile Gly Asn. Glu Ala Glin Lys Gly Asn. Phe Lys Asp Ala 610 615 62O Lieu. Glu Lieu. Lieu. Gly Ala Gly Ile Lieu. Lieu. Glu Phe Glu Pro Glu Lieu. US 2009/01171.57 A1 May 7, 2009 70
- Continued
625 630 635 64 O Lieu. Ile Pro Thr Ile Leu Val Phe Thr Ile Llys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Llys Val Ile Lys Ala Ile Asn. Asn Ala Lieu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Llys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 68O 685 Trp Met Thr Lys Ile Asn Thr Glin Phe Asn Lys Arg Lys Glu Gln Met 690 695 7 OO Tyr Glin Ala Lieu. Glin Asn Glin Val Asn Ala Ile Llys Thir Ile Ile Glu 7 Os 71O 71s 72O Ser Lys Tyr Asn. Ser Tyr Thr Lieu. Glu Glu Lys Asn. Glu Lieu. Thir Asn 72 73 O 73 Llys Tyr Asp Ile Lys Glin Ile Glu Asn. Glu Lieu. Asn Gln Llys Val Ser 740 74. 75O Ile Ala Met Asn. Asn. Ile Asp Arg Phe Lieu. Thr Glu Ser Ser Ile Ser 75s 760 765 Tyr Lieu Met Lys Lieu. Ile Asn. Glu Val Lys Ile Asn Llys Lieu. Arg Glu 770 775 78O Tyr Asp Glu Asn. Wall Lys Thr Tyr Lieu. Lieu. Asn Tyr Ile Ile Glin His 78s 79 O 79. 8OO Gly Ser Ile Leu Gly Glu Ser Glin Gln Glu Lieu. Asn Ser Met Val Thr 805 810 815 Asp Thr Lieu. Asn Asn Ser Ile Pro Phe Llys Leu Ser Ser Tyr Thr Asp 82O 825 830 Asp Llys Ile Lieu. Ile Ser Tyr Phe Asn Llys Phe Phe Lys Arg Ile Llys 835 84 O 845 Ser Ser Ser Val Lieu. Asn Met Arg Tyr Lys Asn Asp Llys Tyr Val Asp 850 855 860 Thir Ser Gly Tyr Asp Ser Asn. Ile Asn. Ile Asin Gly Asp Val Tyr Lys 865 87O 87s 88O Tyr Pro Thr Asn Lys Asn Glin Phe Gly Ile Tyr Asn Asp Llys Lieu. Ser 885 890 895 Glu Val Asn. Ile Ser Glin Asn Asp Tyr Ile Ile Tyr Asp Asn Llys Tyr 9 OO 9 OS 910 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 92O 925 Lys Ile Val Asn. Wall Asn. Asn. Glu Tyr Thir Ile Ile Asn. Cys Met Arg 930 935 94 O Asp Asn. Asn. Ser Gly Trp Llys Val Ser Lieu. Asn His Asn. Glu Ile Ile 945 950 955 96.O Trp. Thir Lieu. Glin Asp Asn Ala Gly Ile Asin Gln Llys Lieu Ala Phe Asn 965 97O 97. Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Llys Trp Ile Phe 98O 985 990 Val Thir Ile Thr Asn Asp Arg Lieu. Gly Asp Ser Llys Lieu. Tyr Ile Asn 995 1 OOO 1 OOS Gly Asn Lieu. Ile Asp Gln Lys Ser Ile Lieu. Asn Lieu. Gly Asn. Ile His 1010 1 O15 1 O2O Val Ser Asp Asn. Ile Lieu. Phe Lys Ile Val Asn. Cys Ser Tyr Thr Arg 1025 103 O 1035 104 O US 2009/01171.57 A1 May 7, 2009 71
- Continued
Cyr Ile Gly Ile Arg Tyr Phe Asn. Ile Phe Asp Llys Glu Lieu. Asp Glu O45 OSO O55 Chir Glu Ile Glin Thr Lieu. Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu O60 O65 Of O Lys Asp Phe Trp Gly Asn Tyr Lieu. Lieu. Tyr Asp Llys Glu Tyr Tyr Lieu. O7s O8O O85 Lieu. Asn Val Lieu Lys Pro Asn. Asn. Phe Ile Asp Arg Arg Lys Asp Ser O90 O95 OO Chir Lieu. Ser Ile Asn. Asn. Ile Arg Ser Thr e Lieu. Lieu Ala Asn Arg OS 10 15 112O Lieu. Tyr Ser Gly Ile Llys Wall Lys Ile Glin Arg Val Asn. Asn. Ser Ser
Thir Asn Asp Asn Lieu Val Arg Lys Asn Asp Glin Val Tyr Ile Asin Phe
Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr
Thir Asn Lys Glu Lys Thir Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe
Asn Glin Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 85 9 O 95 12 OO Phe Lys Asn. Asn. Asn Gly Asn. Asn. Ile Gly Lieu. Lieu. Gly Phe Lys Ala 2O5 21 O 215 Asp Thr Val Val Ala Ser Thir Trp Tyr Tyr Thr His Met Arg Asp His 22O 225 23 O
h r A.S Ser Asn Gly Cys Phe Trp Asin Phe Ile Ser Glu Glu. His Gly 235 24 O 245 rp Glin Glu Lys
<210 SEQ ID NO 6 <211 LENGTH: 1274 &212> TYPE: PRT <213> ORGANISM: Clostridium botulinum Serotype F &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) ... (436) <223> OTHER INFORMATION: Light chain comprising the enzymatic domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (.437) . . . (852) <223> OTHER INFORMATION: Amino-terminal half of heavy chain comprising the translocation domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (853) . . . (1274) <223> OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain.
<4 OO SEQUENCE: 6 Met Pro Val Ala Ile Asn. Ser Phe Asn Tyr Asn Asp Pro Wall Asn Asp 1. 5 1O 15 Asp Thir Ile Leu Tyr Met Glin Ile Pro Tyr Glu Glu Lys Ser Lys Lys 2O 25 3 O Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asin Val Trp Ile Ile Pro Glu 35 4 O 45 Arg Asn. Thir Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser SO 55 6 O Lieu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Lieu. Thir Thr US 2009/01171.57 A1 May 7, 2009 72
- Continued
Asp Ala Glu Lys Asp Arg Tyr Lieu Lys Thir Thir Ile Llys Lieu. Phe Lys 85 9 O 95 Arg Ile Asn. Ser ASn Pro Ala Gly Llys Val Lieu. Lieu. Glin Glu Ile Ser OO OS 1O yr Ala Lys Pro Tyr Lieu. Gly Asn Asp His Thr Pro Ile Asp Glu Phe 15 2O 25 Ser Pro Val Thr Arg Thr Thr Ser Val Asin Ile Llys Lieu Ser Thr Asn 3O 35 4 O Val Glu Ser Ser Met Lieu. Lieu. Asn Lieu. Lieu Val Lieu. Gly Ala Gly Pro 45 SO 55 160 Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Llys Lieu. Ile Asp Pro 65 70 7s Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asin Ile 8O 85 90 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 95 2 OO 2O5 Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210 215 22O Lieu Ala His Glu Lieu. Ile His Ala Lieu. His Gly Lieu. Tyr Gly Ala Arg 225 23 O 235 24 O Gly Val Thr Tyr Glu Glu Thir Ile Glu Val Lys Glin Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg Lieu. Glu Glu Phe Lieu. Thir Phe Gly Gly 26 O 265 27 O Glin Asp Lieu. Asn. Ile Ile Thir Ser Ala Met Lys Glu Lys Ile Tyr Asn 27s 28O 285 Asn Lieu. Lieu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Lieu. Ser Glu Val 290 295 3 OO Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asin Glu Tyr Lys Asp Tyr Phe 3. OS 310 315 32O Glin Trp Llys Tyr Gly Lieu. Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 3.25 330 335 Asn Glu Asn Llys Phe Asn. Glu Ile Tyr Lys Llys Lieu. Tyr Ser Phe Thr 34 O 345 350 Glu Ser Asp Lieu Ala Asn Llys Phe Llys Wall Lys Cys Arg Asn. Thir Tyr 355 360 365 Phe Ile Llys Tyr Glu Phe Lieu Lys Val Pro Asn Lieu. Lieu. Asp Asp Asp 37O 375 38O Ile Tyr Thr Val Ser Glu Gly Phe Asin Ile Gly Asn Lieu Ala Val Asn 385 390 395 4 OO Asn Arg Gly Glin Ser Ile Llys Lieu. Asn Pro Lys Ile Ile Asp Ser Ile 4 OS 410 415 Pro Asp Llys Gly Lieu Val Glu Lys Ile Val Llys Phe Cys Llys Ser Val 42O 425 43 O Ile Pro Arg Lys Gly. Thir Lys Ala Pro Pro Arg Lieu. Cys Ile Arg Val 435 4 4 O 445 Asn Asn Ser Glu Lieu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 45.5 460 Asn Asp Ile Asn Thr Pro Llys Glu Ile Asp Asp Thir Thr Asn Lieu. Asn 465 470 47s 48O US 2009/01171.57 A1 May 7, 2009 73
- Continued
Asn Asn Tyr Arg Asn. Asn Lieu. Asp Glu Val Ile Lieu. Asp Tyr Asn. Ser 485 490 495 Gln Thr Ile Pro Glin Ile Ser Asn Arg Thr Lieu. Asn Thr Lieu Val Glin 5 OO 5 OS 510 Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 52O 525 Glu Glu Tyr Asp Val Val Asp Phe Asin Val Phe Phe Tyr Lieu. His Ala 53 O 535 54 O Gln Llys Val Pro Glu Gly Glu Thir Asn Ile Ser Lieu. Thir Ser Ser Ile 5.45 550 555 560 Asp Thir Ala Lieu. Lieu. Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565 st O sfs Phe Ile Asp Thir Ile Asn Llys Pro Val Asn Ala Ala Lieu. Phe Ile Asp 58O 585 590 Trp Ile Ser Llys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln Lys 595 6 OO 605 Ser Thr Val Asp Llys Ile Ala Asp Ile Ser Lieu. Ile Val Pro Tyr Val 610 615 62O Gly Lieu Ala Lieu. Asn. Ile Ile Ile Glu Ala Glu Lys Gly Asn. Phe Glu 625 630 635 64 O Glu Ala Phe Glu Lieu. Lieu. Gly Val Gly Ile Lieu. Lieu. Glu Phe Val Pro 645 650 655 Glu Lieu. Thir Ile Pro Val Ile Leu Val Phe Thir Ile Llys Ser Tyr Ile 660 665 670 Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn. Asn. Ser 675 68O 685 Lieu. Ile Glu Arg Glu Ala Lys Trip Lys Glu Ile Tyr Ser Trp Ile Val 690 695 7 OO Ser Asn Trp Lieu. Thir Arg Ile Asn Thr Glin Phe Asn Lys Arg Lys Glu 7 Os 71O 71s 72O Glin Met Tyr Glin Ala Lieu. Glin Asn Glin Val Asp Ala Ile Llys Thr Ala 72 73 O 73 Ile Glu Tyr Lys Tyr Asn. Asn Tyr Thir Ser Asp Glu Lys Asn Arg Lieu. 740 74. 75O Glu Ser Glu Tyr Asn. Ile Asn. Asn. Ile Glu Glu Glu Lieu. Asn Llys Llys 75s 760 765 Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 78O Ile Ser Tyr Lieu Met Lys Lieu. Ile Asin Glu Ala Lys Val Gly Lys Lieu. 78s 79 O 79. 8OO Llys Llys Tyr Asp Asn His Val Lys Ser Asp Lieu. Lieu. Asn Tyr Ile Lieu. 805 810 815 Asp His Arg Ser Ile Lieu. Gly Glu Glin Thr Asn. Glu Lieu. Ser Asp Lieu 82O 825 830 Val Thr Ser Thr Lieu. Asn Ser Ser Ile Pro Phe Glu Lieu Ser Ser Tyr 835 84 O 845 Thir Asn Asp Llys Ile Lieu. Ile Ile Tyr Phe Asn Arg Lieu. Tyr Lys Llys 850 855 860 Ile Lys Asp Ser Ser Ile Lieu. Asp Met Arg Tyr Glu Asn. Asn Llys Phe 865 87O 87s 88O US 2009/01171.57 A1 May 7, 2009 74
- Continued Ile Asp Ile Ser Gly Tyr Gly Ser Asn. Ile Ser Ile Asn Gly Asn Val 885 890 895 Tyr Ile Tyr Ser Thr Asn Arg Asn Glin Phe Gly Ile Tyr Asn Ser Arg 9 OO 9 OS 910 Lieu. Ser Glu Val Asn. Ile Ala Glin Asn. Asn Asp Ile Ile Tyr Asn. Ser 915 92O 925 Arg Tyr Glin Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Llys His 930 935 94 O Tyr Llys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile Asin Cys Met 945 950 955 96.O Gly Asn. Asn. Asn. Ser Gly Trp Llys Ile Ser Lieu. Arg Thr Val Arg Asp 965 97O 97. Cys Glu Ile Ile Trp Thir Lieu. Glin Asp Thir Ser Gly Asn Lys Glu Asn 98O 985 990 Lieu. Ile Phe Arg Tyr Glu Glu Lieu. Asn Arg Ile Ser Asn Tyr Ile Asn 995 OOO OOS Llys Trp Ile Phe Val Thir Ile Thr Asn. Asn Arg Lieu. Gly Asn. Ser Arg O1O O15 O2O le Tyr Ile Asin Gly Asn Lieu. Ile Val Glu Lys Ser Ile Ser Asn Lieu O25 O3 O O35 104 O Gly Asp Ile His Val Ser Asp Asn. Ile Lieu. Phe Lys Ile Val Gly Cys O45 OSO O55 Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Llys Val Phe Asn Thr O60 O65 Of O Glu Lieu. Asp Llys Thr Glu Ile Glu Thir Lieu. Tyr Ser Asn. Glu Pro Asp O7s O8O O85 Pro Ser Ile Lieu Lys Asn Tyr Trp Gly Asn Tyr Lieu. Lieu. Tyr Asn Lys
Llys Tyr Tyr Lieu. Phe Asn Lieu. Lieu. Arg Lys Asp Llys Tyr Ile Thr Lieu. OS 10 15 112O Asn Ser Gly Ile Lieu. Asn. Ile Asin Glin Glin Arg Gly Val Thr Glu Gly
Ser Val Phe Lieu. Asn Tyr Lys Lieu. Tyr Glu Gly Val Glu Val Ile Ile
Arg Lys Asn Gly Pro e Asp Ile Ser Asn. Thir Asp Asn. Phe Val Arg
Lys Asn Asp Lieu Ala Tyr Ile Asin Val Val Asp Arg Gly Val Glu Tyr
Arg Lieu. Tyr Ala Asp Thir Lys Ser Glu Lys Glu Lys Ile Ile Arg Thr 85 9 O 95 12 OO Ser Asn Lieu. Asn Asp Ser Lieu. Gly Glin Ile e Val Met Asp Ser Ile
Gly Asn. Asn. Cys Thr Met Asn. Phe Glin Asn. Asn. Asn Gly Ser Asn. Ile
Gly Lieu. Lieu. Gly Phe His Ser Asn. Asn Lieu Val Ala Ser Ser Trp Tyr
yr Asn. Asn. Ile Arg Arg Asn. Thir Ser Ser Asn Gly Cys Phe Trp Ser
Ser Ile Ser Lys Glu Asn Gly Trip Lys Glu US 2009/01171.57 A1 May 7, 2009 75
- Continued <210 SEQ ID NO 7 <211 LENGTH: 1297 &212> TYPE: PRT <213> ORGANISM: Clostridium botulinum Serotype G &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) ... (44.2) <223> OTHER INFORMATION: Light chain comprising the enzymatic domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (443) . . . (852) <223> OTHER INFORMATION: Amino-terminal half of heavy chain comprising the translocation domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (853) . . . (1297) <223> OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain.
<4 OO SEQUENCE: 7 Met Pro Val Asn. Ile Lys Asn. Phe Asn Tyr Asn Asp Pro Ile Asn. Asn 1. 5 1O 15 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 2O 25 3 O Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 4 O 45 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Glin Phe Asn Ala Ser Thr Gly SO 55 6 O Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Lieu Lys 65 70 7s 8O Thir Asp Ala Glu Lys Asp Llys Phe Lieu Lys Thr Met Ile Llys Lieu. Phe
Asn Arg Ile Asn. Ser Llys Pro Ser Gly Glin Arg Lieu. Lieu. Asp Met Ile OO OS 1O Val Asp Ala Ile Pro Tyr Lieu. Gly Asn Ala Ser Thr Pro Pro Asp Llys 15 2O 25 Phe Ala Ala Asn. Wall Ala Asn Val Ser Ile Asn Llys Lys Ile Ile Glin 3O 35 4 O Pro Gly Ala Glu Asp Glin Ile Lys Gly Lieu Met Thr Asn Lieu. Ile Ile 45 SO 55 160 Phe Gly Pro Gly Pro Val Lieu Ser Asp Asin Phe Thr Asp Ser Met Ile 65 70 7s Met Asin Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 8O 85 90 le Arg Phe Cys Pro Ser Cys Lieu. Asn Val Phe Asn Asn Val Glin Glu 95 2 OO 2O5 Asn Lys Asp Thir Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 22O Ala Lieu. Thir Lieu Met His Glu Lieu. Ile His Val Lieu. His Gly Lieu. Tyr 225 23 O 235 24 O Gly Ile Lys Ile Ser Asn Lieu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 Phe Met Gln His Ser Asp Pro Val Glin Ala Glu Glu Lieu. Tyr Thr Phe 26 O 265 27 O Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asin Ile 27s 28O 285 Tyr Asn Lys Ala Lieu. Glin Asn. Phe Glin Asp Ile Ala Asn Arg Lieu. Asn 290 295 3 OO US 2009/01171.57 A1 May 7, 2009 76
- Continued Ile Val Ser Ser Ala Glin Gly Ser Gly Ile Asp Ile Ser Lieu. Tyr Lys 3. OS 310 315 32O Glin Ile Tyr Lys Asn Llys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 3.25 330 335 Tyr Ser Val Asp Lys Asp Llys Phe Asp Llys Lieu. Tyr Lys Ala Lieu Met 34 O 345 350 Phe Gly Phe Thr Glu Thir Asn Lieu Ala Gly Glu Tyr Gly Ile Llys Thr 355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Lieu Pro Pro Ile Llys Thr Glu Lys 37O 375 38O Lieu. Lieu. Asp Asn. Thir Ile Tyr Thr Glin Asn. Glu Gly Phe Asn. Ile Ala 385 390 395 4 OO Ser Lys Asn Lieu Lys Thr Glu Phe Asin Gly Glin Asn Lys Ala Val Asn 4 OS 410 415 Lys Glu Ala Tyr Glu Glu Ile Ser Lieu. Glu. His Lieu Val Ile Tyr Arg 42O 425 43 O Ile Ala Met Cys Llys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 4 4 O 445 Glin Cys Ile Ile Val Asn. Asn. Glu Asp Lieu. Phe Phe Ile Ala Asn Lys 450 45.5 460 Asp Ser Phe Ser Lys Asp Lieu Ala Lys Ala Glu Thir Ile Ala Tyr Asn 465 470 47s 48O Thr Glin Asn. Asn. Thir Ile Glu Asn. Asn. Phe Ser Ile Asp Gln Lieu. Ile 485 490 495 Lieu. Asp Asn Asp Lieu. Ser Ser Gly Ile Asp Lieu Pro Asn. Glu Asn Thr 5 OO 5 OS 510 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Llys 515 52O 525 Glin Ser Ala Lieu Lys Lys Ile Phe Val Asp Gly Asp Ser Lieu. Phe Glu 53 O 535 54 O Tyr Lieu. His Ala Glin Thr Phe Pro Ser Asn Ile Glu Asn Lieu Gln Leu 5.45 550 555 560 Thir Asn. Ser Lieu. Asn Asp Ala Lieu. Arg Asn. Asn. Asn Llys Val Tyr Thr 565 st O sfs Phe Phe Ser Thr Asn Lieu Val Glu Lys Ala Asn Thr Val Val Gly Ala 58O 585 590 Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thir Ser 595 6 OO 605 Glu Ser Thr Gln Lys Ser Thr Ile Asp Llys Val Ser Asp Val Ser Ile 610 615 62O Ile Ile Pro Tyr Ile Gly Pro Ala Lieu. Asn Val Gly Asn Glu. Thir Ala 625 630 635 64 O Lys Glu Asn. Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Lieu. 645 650 655 Met Glu Phe Ile Pro Glu Lieu. Ile Val Pro Ile Val Gly Phe Phe Thr 660 665 670 Lieu. Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser 675 68O 685 Asn Ala Lieu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Lieu 690 695 7 OO Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Glin Phe Tyr Thr Ile US 2009/01171.57 A1 May 7, 2009 77
- Continued
Lys Glu Arg Met Tyr Asn Ala Lieu. Asn. Asn Glin Ser Glin Ala Ile Glu 72 73 O 73 Lys Ile Ile Glu Asp Glin Tyr Asn Arg Tyr Ser Glu Glu Asp Llys Met 740 74. 75O Asn. Ile Asn. Ile Asp Phe Asn Asp Ile Asp Phe Llys Lieu. Asn Glin Ser 75s 760 765 Ile Asn Lieu Ala Ile Asn. Asn. Ile Asp Asp Phe Ile Asn Glin Cys Ser 770 775 78O Ile Ser Tyr Lieu Met Asn Arg Met Ile Pro Lieu Ala Val Lys Llys Lieu. 78s 79 O 79. 8OO Lys Asp Phe Asp Asp Asn Lieu Lys Arg Asp Lieu. Lieu. Glu Tyr Ile Asp 805 810 815 Thir Asn. Glu Lieu. Tyr Lieu. Lieu. Asp Glu Val Asn. Ile Lieu Lys Ser Lys 82O 825 830 Val Asn Arg His Lieu Lys Asp Ser Ile Pro Phe Asp Lieu. Ser Lieu. Tyr 835 84 O 845 Thr Lys Asp Thir Ile Lieu. Ile Glin Val Phe Asn Asn Tyr Ile Ser Asn 850 855 860 Ile Ser Ser Asn Ala Ile Lieu. Ser Lieu. Ser Tyr Arg Gly Gly Arg Lieu. 865 87O 87s 88O Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 890 895 Ile Phe Asn Asp Ile Gly Asn Gly Glin Phe Llys Lieu. Asn. Asn. Ser Glu 9 OO 9 OS 910 Asn Ser Asn Ile Thr Ala His Glin Ser Llys Phe Val Val Tyr Asp Ser 915 92O 925 Met Phe Asp Asin Phe Ser Ile Asin Phe Trp Val Arg Thr Pro Llys Tyr 930 935 94 O Asn Asn. Asn Asp Ile Glin Thr Tyr Lieu. Glin Asn. Glu Tyr Thir Ile Ile 945 950 955 96.O Ser Cys Ile Lys Asn Asp Ser Gly Trp Llys Val Ser Ile Lys Gly Asn 965 97O 97. Arg Ile Ile Trp Thir Lieu. Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 98O 985 990 Phe Phe Glu Tyr Ser Ile Lys Asp Asn. Ile Ser Asp Tyr Ile Asn Lys 995 OOO OOS rp Phe Ser Ile Thir Ile Thr Asn Asp Arg Lieu. Gly Asn Ala Asn. Ile
yr Ile Asin Gly Ser Lieu Lys Llys Ser Glu Lys Ile Lieu. Asn Lieu. Asp O25 O3 O O35 104 O Arg Ile Asn. Ser Ser Asn Asp Ile Asp Phe Llys Lieu. Ile Asn. Cys Thr
Asp Thir Thr Llys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg
Glu Lieu. Asn Ala Thr Glu Val Ser Ser Lieu. Tyr Trp Ile Glin Ser Ser
h r A.S Thir Lieu Lys Asp Phe Trp Gly Asn Pro Lieu. Arg Tyr Asp Thr
Gln Tyr Tyr Lieu Phe Asn Glin Gly Met Glin Asn Ile Tyr Ile Llys Tyr 105 11 O 115 112O US 2009/01171.57 A1 May 7, 2009 78
- Continued
Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn 25 13 O 135
Asn Ala Ala Ile ASn Tyr Glin Asn Lieu. Tyr Lel Gly Lell Arg Phe Ile 4 O 145 15 O
e Llys Lys Ala Ser Asn. Ser Arg Asn Ile Asp Asn Ile Wall 55 16 O
Arg Glu Gly Asp Tyr Ile Tyr Lieu. Asn Ile Ile Ser Asp Glu 70 17s
Ser Tyr Arg Val Tyr Val Lieu Val Asn Ser Ile Glin Thr Gin 85 19 O 12 OO
Lieu. Phe Lieu Ala Pro Ile Asn Asp Asp Pro h r Asp Wall Lieu 2O5 21 O
Glin Ile Llys Llys Tyr Tyr Glu Lys Thr Thir Glin Ile Lieu. 22O 225
Cys Glu Lys Asp Thr Lys Thr Phe Gly Luell Ile Gly Llys Phe 235 24 O
Val Lys Asp Tyr Gly Tyr Val Trp Asp Thir Asn Phe Cys 250 255
le Ser Glin Trp Tyr Lieu. Arg Arg Ile Ser Ile Asn Llys Lieu. 265 27 O 128O
Arg Lieu. Gly Cys Asn Trp Glin Phe Ile Pro Glu Gly Trp Thr 285 29 O
Glu
<210 SEQ ID NO 8 <211 LENGTH: 1315 &212> TYPE: PRT <213> ORGANISM: Clostridium tetani &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) ... (441) <223> OTHER INFORMATION: Light chain comprising the enzymatic domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (442) ... (870) <223> OTHER INFORMATION: Amino-terminal half of heavy chain comprising the translocation domain. <221 NAME/KEY: DOMAIN <222> LOCATION: (871) . . . (1315) <223> OTHER INFORMATION: Carboxyl-terminal half of heavy chain comprising the binding domain.
<4 OO SEQUENCE: 8
Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Wall Asn. Asn 1. 5 15
Asp Thir Ile Ile Met Met Glu Pro Pro Tyr Cys Gly Luell Asp Ile 2O 25 3 O
Tyr Ala Phe Lys Ile Thir Asp Arg Ile Trp Ile Wall Pro Glu 35 4 O 45
Arg Glu Phe Gly Thir Pro Glu Asp Phe Asn Pro Pro Ser Ser SO 55 6 O
Lell Ile Glu Gly Ala Ser Glu Asp Pro Asn Luell Arg Thr 65 70 7s
Asp Ser Asp Asp Arg Phe Luell Glin Thir Met Wall Luell Phe Asn 85 9 O 95
Arg Ile Asn Asn Wall Ala Gly Glu Ala Luell Lell Asp Ile Ile 1 OO 105 11O US 2009/01171.57 A1 May 7, 2009 79
- Continued
Ala Ile Pro Tyr Lell Gly Asn Ser Ser Lell Lell Asp Lys Phe 12O 125
Thir Asn Ser Asn Ser Wall Ser Phe Asn Luell Lell Glu Glin Asp Pro 135 14 O
Gly Ala Thir Thir Lys Ser Ala Met Luell Thir Asn Lell Ile Ile Phe 150 155 160
Pro Gly Pro Wall Lell Asn Asn Glu Wall Arg Gly Ile Wall Luell 17O 17s
Wall Asp Asn Lys Asn Phe Pro Arg Asp Gly Phe Gly Ser 185 190
le Met Glin Met Ala Phe Pro Glu Wall Pro Thir Phe Asp Asn 95 2 OO 2O5
Wall Ile Glu Asn Ile Thir Ser Luell Thir Ile Gly Ser Phe 210 215 22O
Glin Asp Pro Ala Lell Lell Lell Met His Glu Luell Ile His Wall Luell His 225 23 O 235 24 O
Gly Luell Gly Met Glin Wall Ser Ser His Glu Ile Ile Pro Ser 245 250 255
Glin Glu Ile Met Glin His Thir Pro Ile Ser Ala Glu Glu Luell 26 O 265 27 O
Phe Thir Phe Gly Glin Asp Ala Asn Luell Ile Ser Ile Asp Ile 27s 28O 285
Asn Asp Luell Glu Lys Thir Luell Asn Asp Tyr Ala Ile Ala Asn 290 295 3 OO
Lys Luell Ser Glin Wall Thir Ser Asn Asp Pro Asn Ile Asp Ile Asp 3. OS 310 315
Ser Glin Ile Tyr Glin Glin Glin Phe Asp Asp Ser 3.25 330 335
Asn Gly Glin Ile Wall Asn Glu Asp Phe Glin Ile Luell Asn 34 O 345 350
Ser Ile Met Gly Phe Thir Glu Ile Glu Luell Gly Phe Asn 355 360 365
Ile Thir Arg Lell Ser Phe Ser Met ASn His Asp Pro Wall 37O 375 38O
Ile Pro Asn Luell Lell Asp Asp Thir Ile ASn Asp Thir Glu Gly Phe 385 390 395 4 OO
Asn Ile Glu Ser Lys Asp Lell Ser Glu Tyr Gly Glin Asn Met 4 OS 410 415
Arg Wall Asn Thir Asn Ala Phe Asn Wall Asp Gly Ser Gly Luell Wall 42O 425 43 O
Ser Luell Ile Gly Lell Ile Ile Pro Pro Thir Asn Ile 435 4 4 O 445
Arg Glu Asn Luell Tyr Asn Thir Ala Ser Luell Thir Asp Luell Gly Gly 450 45.5 460
Glu Luell Ile Lys Ile Asn Glu Asp Luell Thir Phe Ile Ala Glu 465 470 47s
Lys Asn Ser Phe Ser Glu Glu Pro Phe Glin Asp Glu Ile Wall Ser Tyr 485 490 495
Asn Thir Asn Lys Pro Lell Asn Phe Asn Tyr Ser Lell Asp Ile 5 OO 5 OS 510 US 2009/01171.57 A1 May 7, 2009 80
- Continued
Ile Wall Asp Asn Lell Glin Ser Ile Thir Lell Pro Asn Asp Arg 515 52O 525
Thir Thir Pro Wall Thir Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Llys Ser 53 O 535 54 O
Asn Ala Ala Ser Thir Ile Glu Ile His Asn Ile Asp Asp Asn Thir Ile 5.45 550 555 560
Tyr Glin Luell Tyr Ala Glin Ser Pro Thir Thir Lell Glin Arg Ile 565 st O sfs
Thir Met Thir Asn Ser Wall Asp Asp Ala Luell Ile Asn Ser Thir Lys Ile 58O 585 590
Tyr Ser Phe Pro Ser Wall Ile Ser Wall Asn Glin Gly Ala Glin 595 6 OO 605
Gly Ile Luell Phe Lell Glin Trp Wall Arg Asp Ile Ile Asp Asp Phe Thr 610 615 62O
Asn Glu Ser Ser Glin Lys Thir Thir Ile Asp Lys Ile Ser Asp Wall Ser 625 630 635 64 O
Thir Ile Wall Pro Tyr Ile Gly Pro Ala Luell ASn Ile Wall Gln Gly 645 650 655
Tyr Glu Gly Asn Phe Ile Gly Ala Luell Glu Thir Thir Gly Wall Wall Lieu 660 665 670
Lell Luell Glu Ile Pro Glu Ile Thir Luell Pro Wall Ile Ala Ala Lieu 675 68O 685
Ser Ile Ala Glu Ser Ser Thir Glin Glu Lys Ile Ile Thir Ile 690 695 7 OO
Asp Asn Phe Luell Glu Arg Glu Trp Ile Glu Wall 7 Os 71s
Lell Wall Ala Lys Trp Lell Gly Thir Wall ASn Thir Glin Phe Gln Lys 72 73 O 73
Arg Ser Glin Met Tyr Arg Ser Luell Glu Glin Wall Asp Ala Ile 740 74.
Lys Ile Ile Asp Tyr Glu Ile Tyr Ser Gly Pro Asp Llys 75s 760 765
Glu Glin Ile Ala Asp Glu Ile Asn Asn Luell Asn Luell Glu Glu 770 775
Lys Ala Asn Ala Met Ile Asn Ile Asn Phe Met Arg Glu Ser 78s 79 O
Ser Arg Ser Phe Lell Wall Asn Glin Met Ile Glu Ala Lys Glin 805 810
Lell Luell Glu Phe Asp Thir Glin Ser Asn Lell Met Glin Tyr Ile 82O 825
Lys Ala Asn Ser Lys Phe Ile Gly Ile Thir Lell Lieu. Glu 835 84 O
Ser Ile Asn Lys Wall Phe Ser Thir Pro Pro Phe Ser Tyr Ser 850 855
Lys Asn Luell Asp Cys Wall Asp Asn Glu Asp Ile Asp Wall Ile 865
Lell Ser Thir Ile Lell Asn Luell Asp Asn Asn Asp Ile Ile 885 890
Ser Asp Ile Ser Gly Phe Asn Ser Ser Wall Thir Pro Asp Ala 9 OO 9 OS
Glin Luell Wall Pro Gly Ile Asn Gly Ala His Lell Wall Asn. Asn US 2009/01171.57 A1 May 7, 2009 81
- Continued
915 92O 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 94 O Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Lieu. Arg Val Pro Llys 945 950 955 96.O Val Ser Ala Ser His Lieu. Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 97O 97. Ile Ser Ser Met Lys Llys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 98O 985 990 Val Ser Lieu Lys Gly Asn. Asn Lieu. Ile Trp Thir Lieu Lys Asp Ser Ala 995 OOO OOS Gly Glu Val Arg Glin Ile Thr Phe Arg Asp Lieu Pro Asp Llys Phe Asn
Ala Tyr Lieu Ala Asn Llys Trp Val Phe Ile Thir Ile Thr Asn Asp Arg O25 O3 O O35 104 O Lieu. Ser Ser Ala Asn Lieu. Tyr Ile Asin Gly Val Lieu Met Gly Ser Ala
Glu Ile Thr Gly Lieu. Gly Ala Ile Arg Glu Asp Asn. Asn. Ile Thr Lieu.
Llys Lieu. Asp Arg Cys Asn. Asn. Asn. Asn Glin Tyr Val Ser Ile Asp Llys
Phe Arg Ile Phe Cys Lys Ala Lieu. ASn Pro Lys Glu. Ile Glu Lys Lieu.
yr Thr Ser Tyr Lieu Ser Ile Thr Phe Lieu. Arg Asp Phe Trp Gly Asn OS 10 15 112O
Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Lieu. e Pro Wall Ala Ser Ser
Ser Lys Asp Val Glin Lieu Lys Asn. Ile Thr Asp Tyr Met Tyr Lieu. Thir
Asn Ala Pro Ser Tyr Thr Asn Gly Lys Lieu. Asn. Ile Tyr Tyr Arg Arg
Lieu. Tyr Asn Gly Lieu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn. Asn
Glu Ile Asp Ser Phe Wall Lys Ser Gly Asp Phe Ile Llys Lieu. Tyr Val 85 9 O 95 12 OO Ser Tyr Asn. Asn. Asn. Glu. His Ile Val Gly Tyr Pro Lys Asp Gly Asn 2O5 21 O 215 Ala Phe Asn. Asn Lieu. Asp Arg Ile Lieu. Arg Val Gly Tyr Asn Ala Pro 22O 225 23 O Gly Ile Pro Lieu. Tyr Llys Llys Met Glu Ala Val Llys Lieu. Arg Asp Lieu.
Llys Thr Tyr Ser Val Glin Lieu Lys Lieu. Tyr Asp Asp Lys Asn Ala Ser 250 255 26 O Lieu. Gly Lieu Val Gly Thr His Asn Gly Glin Ile Gly Asn Asp Pro Asn 265 27 O 27s 128O Arg Asp Ile Lieu. Ile Ala Ser Asn Trp Tyr Phe Asn His Lieu Lys Asp 285 29 O 295 Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp 3OO 305 31 O
h r A.S Asp US 2009/01171.57 A1 May 7, 2009 82
- Continued
<210 SEQ ID NO 9 <211 LENGTH: 1268 &212> TYPE: PRT <213> ORGANISM: Clostridium baratii
<4 OO SEQUENCE: 9 Met Pro Val Asn. Ile Asn. Asn. Phe Asn Tyr Asn Asp Pro Ile Asn. Asn 1. 5 1O 15 Thir Thir Ile Leu Tyr Met Lys Met Pro Tyr Tyr Glu Asp Ser Asn Lys 2O 25 3 O Tyr Tyr Lys Ala Phe Glu Ile Met Asp Asin Val Trp Ile Ile Pro Glu 35 4 O 45 Arg Asn Ile Ile Gly Lys Llys Pro Ser Asp Phe Tyr Pro Pro Ile Ser SO 55 6 O Lieu. Asp Ser Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Lieu. Thir Thr 65 70 7s 8O Asp Ala Glu Lys Asp Arg Phe Lieu Lys Thr Val Ile Llys Lieu. Phe Asn 85 9 O 95 Arg Ile Asn. Ser ASn Pro Ala Gly Glin Val Lieu. Lieu. Glu Glu Ile Llys OO OS 1O Asn Gly Llys Pro Tyr Lieu. Gly Asn Asp His Thr Ala Val Asn. Glu Phe 15 2O 25 Cys Ala Asn. Asn Arg Ser Thr Ser Val Glu Ile Lys Glu Ser Asn Gly 3O 35 4 O Thir Thr Asp Ser Met Leu Lleu. Asn Lieu Val Ile Leu Gly Pro Gly Pro 45 SO 55 160 Asn Ile Leu Glu. Cys Ser Thr Phe Pro Val Arg Ile Phe Pro Asn Asn 65 70 7s le Ala Tyr Asp Pro Ser Glu Lys Gly Phe Gly Ser Ile Gln Leu Met 8O 85 90 Ser Phe Ser Thr Glu Tyr Glu Tyr Ala Phe Asn Asp Asn Thr Asp Leu 95 2 OO 2O5 Phe Ile Ala Asp Pro Ala Ile Ser Lieu Ala His Glu Lieu. Ile His Val 210 215 22O Lieu. His Gly Lieu. Tyr Gly Ala Lys Gly Val Thr Asn Llys Llys Val Ile 225 23 O 235 24 O Glu Val Asp Glin Gly Ala Lieu Met Ala Ala Glu Lys Asp Ile Lys Ile 245 250 255 Glu Glu Phe Ile Thr Phe Gly Gly Glin Asp Lieu. Asn Ile Ile Thr Asn 26 O 265 27 O Ser Thr Asn Gln Lys Ile Tyr Val Ile Leu Lleu Ser Asn Tyr Thr Ala 27s 28O 285 Ile Ala Ser Arg Lieu. Ser Glin Val Asn Arg Asn. Asn. Ser Ala Lieu. Asn 290 295 3 OO Thir Thr Tyr Tyr Lys Asn Phe Phe Gln Trp Llys Tyr Gly Lieu. Asp Glin 3. OS 310 315 32O Asp Ser Asn Gly Asn Tyr Thr Val Asn. Ile Ser Llys Phe Asn Ala Ile 3.25 330 335 Tyr Lys Llys Lieu. Phe Ser Phe Thr Glu. Cys Asp Lieu Ala Glin Llys Phe 34 O 345 350 Glin Val Lys Asn Arg Ser Asn Tyr Lieu Phe His Phe Llys Pro Phe Arg US 2009/01171.57 A1 May 7, 2009 83
- Continued
355 360 365 Lieu. Lieu. Asp Lieu. Lieu. Asp Asp Asn. Ile Tyr Ser Ile Ser Glu Gly Phe 37O 375 38O Asn. Ile Gly Ser Lieu. Arg Val Asn. Asn. Asn Gly Glin Asn. Ile Asn Lieu 385 390 395 4 OO Asn Ser Arg Ile Val Gly Pro Ile Pro Asp Asn Gly Lieu Val Glu Arg 4 OS 410 415 Phe Val Gly Lieu. Cys Llys Ser Ile Val Ser Lys Lys Gly. Thir Lys Asn 42O 425 43 O Ser Lieu. Cys Ile Llys Val Asn. Asn Arg Asp Lieu. Phe Phe Val Ala Ser 435 4 4 O 445 Glu Ser Ser Tyr Asn. Glu Asn Gly Ile Asn. Ser Pro Llys Glu Ile Asp 450 45.5 460 Asp Thir Thir Ile Thr Asn. Asn. Asn Tyr Lys Lys Asn Lieu. Asp Glu Val 465 470 47s 48O Ile Lieu. Asp Tyr Asn. Ser Asp Ala Ile Pro Asn Lieu. Ser Ser Arg Lieu. 485 490 495 Lieu. Asn. Thir Thir Ala Glin Asn Asp Ser Tyr Val Pro Llys Tyr Asp Ser 5 OO 5 OS 510 Asn Gly. Thir Ser Glu Ile Lys Glu Tyr Thr Val Asp Llys Lieu. Asn Val 515 52O 525 Phe Phe Tyr Lieu. Tyr Ala Gln Lys Ala Pro Glu Gly Glu Ser Ala Ile 53 O 535 54 O Ser Lieu. Thir Ser Ser Val Asn. Thir Ala Lieu. Lieu. Asp Ala Ser Llys Val 5.45 550 555 560 Tyr Thr Phe Phe Ser Ser Asp Phe Ile Asn Thr Val Asn Llys Pro Val 565 st O sfs Glin Ala Ala Lieu. Phe Ile Ser Trp Ile Glin Glin Val Ile Asin Asp Phe 58O 585 590 Thir Thr Glu Ala Thr Glin Llys Ser Thir Ile Asp Llys Ile Ala Asp Ile 595 6 OO 605 Ser Lieu. Ile Val Pro Tyr Val Gly Lieu Ala Lieu. Asn. Ile Gly Asn. Glu 610 615 62O Val Glin Lys Gly Asn. Phe Lys Glu Ala Ile Glu Lieu. Lieu. Gly Ala Gly 625 630 635 64 O
Ile Lleu Lleu. Glu Phe Wall Pro Glu Lieu Lleu. Ile Pro Thir Ile Lieu. Wall 645 650 655 Phe Thir Ile Llys Ser Phe Ile Asin Ser Asp Asp Ser Lys Asn Lys Ile 660 665 670 Ile Lys Ala Ile Asn. Asn Ala Lieu. Arg Glu Arg Glu Lieu Lys Trp Llys 675 68O 685 Glu Val Tyr Ser Trp Ile Val Ser Asn Trp Lieu. Thr Arg Ile Asn Thr 690 695 7 OO Glin Phe Asn Lys Arg Lys Glu Gln Met Tyr Glin Ala Lieu. Glin Asn Glin 7 Os 71O 71s 72O Val Asp Gly Ile Llys Lys Ile Ile Glu Tyr Lys Tyr Asn. Asn Tyr Thr 72 73 O 73 Lieu. Asp Glu Lys Asn Arg Lieu. Arg Ala Glu Tyr Asn. Ile Tyr Ser Ile 740 74. 75O Lys Glu Glu Lieu. Asn Llys Llys Val Ser Lieu Ala Met Glin Asn. Ile Asp 75s 760 765 US 2009/01171.57 A1 May 7, 2009 84
- Continued
Arg Phe Lieu. Thr Glu Ser Ser Ile Ser Tyr Lieu Met Lys Lieu. Ile Asn 770 775 78O Glu Ala Lys Ile Asn Llys Lieu. Ser Glu Tyr Asp Lys Arg Val Asn Glin 78s 79 O 79. 8OO Tyr Lieu. Lieu. Asn Tyr Ile Lieu. Glu Asn Ser Ser Thr Lieu. Gly. Thir Ser 805 810 815
Ser Wall Pro Glu Lieu. Asn. Asn Lieu Wal Ser Asn. Thir Lieu. Asn. Asn. Ser 82O 825 830 Ile Pro Phe Glu Lieu Ser Glu Tyr Thr Asn Asp Lys Ile Lieu. Ile His 835 84 O 845 Ile Lieu. Ile Arg Phe Tyr Lys Arg Ile Ile Asp Ser Ser Ile Lieu. Asn 850 855 860 Met Lys Tyr Glu Asn Asn Arg Phe Ile Asp Ser Ser Gly Tyr Gly Ser 865 87O 87s 88O Asn. Ile Ser Ile Asn Gly Asp Ile Tyr Ile Tyr Ser Thr Asn Arg Asn 885 890 895 Glin Phe Gly Ile Tyr Ser Ser Arg Lieu Ser Glu Val Asn Ile Thr Glin 9 OO 9 OS 910 Asn Asn. Thir Ile Ile Tyr Asn Ser Arg Tyr Glin Asn Phe Ser Val Ser 915 92O 925 Phe Trp Val Arg Ile Pro Llys Tyr Asn. Asn Lieu Lys Asn Lieu. Asn. Asn 930 935 94 O Glu Tyr Thir Ile Ile Asn. Cys Met Arg Asn. Asn. Asn. Ser Gly Trp Llys 945 950 955 96.O Ile Ser Lieu. Asn Tyr Asn Asn Ile Ile Trp Thr Lieu. Glin Asp Thir Thr 965 97O 97. Gly Asn. Asn Glin Llys Lieu Val Phe Asn Tyr Thr Glin Met Ile Asp Ile 98O 985 990 Ser Asp Tyr Ile Asn Lys Trp Thr Phe Val Thr Ile Thr Asn Asn Arg 995 OOO OOS Lieu. Gly His Ser Llys Lieu. Tyr Ile Asin Gly Asn Lieu. Thir Asp Glin Lys O1O O15 O2O Ser Ile Lieu. Asn Lieu. Gly Asn. Ile His Val Asp Asp Asn. Ile Lieu. Phe O25 O3 O O35 104 O Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe
Lys Ile Phe Asn Met Glu Lieu. Asp Llys Thr Glu Ile Glu Thir Lieu. Tyr
His Ser Glu Pro Asp Ser Thr Ile Leu Lys Asp Phe Trp Gly Asn Tyr
Lieu. Lieu. Tyr Asn Llys Llys Tyr Tyr Lieu. Lieu. Asn Lieu Lleu Lys Pro Asn
Met Ser Val Thir Lys Asn. Ser Asp Ile Lieu. Asn. Ile Asn Arg Glin Arg 105 11 O 115 112O Gly Ile Tyr Ser Lys Thr Asn Ile Phe Ser Asn Ala Arg Lieu. Tyr Thr 125 13 O 135 Gly Val Glu Val Ile Ile Arg Llys Val Gly Ser Thr Asp Thir Ser Asn
Chir Asp Asin Phe Val Arg Lys Asn Asp Thr Val Tyr Ile Asin Val Val US 2009/01171.57 A1 May 7, 2009 85
- Continued
Asn Ser Glu Luell Tyr Ala Asp Wal Ser Thir Ser Ala 18O
Thir Ile Arg Arg Ile Ser Asn Ser Asn Tyr Asn 195 12 OO
Glin Met Ile Asp Ser Asn Thir Met
Thir Asn Asn Asp Lell Gly Phe His
Asn Lieu Wall Ser Trp Cyr Lys Asn Ile Arg Asn 245
Arg Asn. Asn Phe Trp Ser Phe Ile Ser Glu His 26 O
Glin Glu
<210 SEQ ID NO 10 <211 LENGTH: 1251 &212> TYPE : PRT <213> ORGANISM: Clostridium butyricum
<4 OO SEQUENCE: 10
Met Pro Thir Ile Asn Ser Phe Asn Asn Asp Pro Wall Asn Asn Arg 1. 5 15
Thir Ile Luell Tyr Ile Lys Pro Gly Gly Glin Glin Phe Ser 2O 25 3 O
Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Wall Ile 35 4 O 45
Gly Thir Ile Pro Glin Asp Phe Luell Pro Pro Thir Ser Lell Asn Gly SO 55 6 O
Asp Ser Ser Asp Pro Asn Luell Glin Ser Asp Glin Glu Lys 65 70 7s
Asp Phe Lieu Lys le Wall Thir Ile Asn Arg Ile Asn Asp 85
Asn Luell Ser Gly Arg le Lell Luell Glu Glu Ser Ala Asn Pro OO OS
yr Luell Gly Asn Asp Asn Thir Pro Asp Gly Phe Ile Ile Asn Asp 15 2O
Ala Ser Ala Wall Pro le Glin Phe Ser Asn Ser Glin Ser Ile Luell 3O 35
Lell Pro Asn Wall Ile le Met Gly Ala Glu Asp Lell Phe Glu Thir 45 SO 160
Asn Ser Ser ASn Ile Lell Arg Asn Asn Met Pro Ser Asn His 65 70
Phe Gly Ser Ile Ala Ile Wall Thir Phe Pro Glu Ser Phe 85
Phe Asp Asn Ser Met Asn Glu Phe le Glin Asp Pro Ala Luell 2 OO 2O5
Luell Met His Glu. Lell Ile His Ser Luell His Gly Lell Gly Ala 210 215 22O
Lys Gly Ile Th Thir Lys Thir Ile Thir Glin Glin Asn Pro Luell 225 23 O 235 24 O
Ile Thir Asn Ile Arg Gly Thir Asn Ile Glu Glu Phe Lell Thir Phe Gly 245 250 255 US 2009/01171.57 A1 May 7, 2009 86
- Continued
Gly. Thir Asp Lieu. Asn. Ile Ile Thir Ser Ala Glin Ser Asn Asp Ile Tyr 26 O 265 27 O Thir Asn Lieu. Lieu Ala Asp Tyr Lys Lys Ile Ala Ser Llys Lieu. Ser Lys 27s 28O 285 Val Glin Val Ser ASn Pro Lieu. Lieu. Asn Pro Tyr Lys Asp Val Phe Glu 290 295 3 OO Ala Lys Tyr Gly Lieu. Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 3. OS 310 315 32O Ile Asn Llys Phe Asn Asp Ile Phe Llys Llys Lieu. Tyr Ser Phe Thr Glu 3.25 330 335 Phe Asp Leu Ala Thr Lys Phe Glin Val Lys Cys Arg Glin Thr Tyr Ile 34 O 345 350 Gly Glin Tyr Lys Tyr Phe Llys Lieu. Ser Asn Lieu. Lieu. Asn Asp Ser Ile 355 360 365 Tyr Asn. Ile Ser Glu Gly Tyr Asn. Ile Asn. Asn Lieu Lys Val Asn. Phe 37O 375 38O Arg Gly Glin Asn Ala Asn Lieu. Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 4 OO Gly Arg Gly Lieu Val Llys Lys Ile Ile Arg Phe Cys Lys Asn. Ile Val 4 OS 410 415 Ser Val Lys Gly Ile Arg Llys Ser Ile Cys Ile Glu Ile Asin Asn Gly 42O 425 43 O Glu Lieu. Phe Phe Val Ala Ser Glu Asn. Ser Tyr Asn Asp Asp Asn. Ile 435 4 4 O 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thir Ser Asn Asn Asn Tyr 450 45.5 460 Glu Asn Asp Lieu. Asp Glin Val Ile Lieu. Asn. Phe Asn. Ser Glu Ser Ala 465 470 47s 48O Pro Gly Lieu. Ser Asp Glu Lys Lieu. Asn Lieu. Thir Ile Glin Asn Asp Ala 485 490 495 Tyr Ile Pro Llys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 5 OO 5 OS 510 Asp Wall Asn. Glu Lieu. Asn Val Phe Phe Tyr Lieu. Asp Ala Glin Llys Val 515 52O 525 Pro Glu Gly Glu Asn. Asn. Wall Asn Lieu. Thir Ser Ser Ile Asp Thir Ala 53 O 535 54 O Lieu. Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 5.45 550 555 560 Asn Asn Val Asn Llys Pro Val Glin Ala Ala Lieu. Phe Val Gly Trp Ile 565 st O sfs Gln Glin Val Lieu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 58O 585 590 Val Asp Llys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Lieu 595 6 OO 605 Ala Lieu. Asn. Ile Gly Asn. Glu Ala Glin Lys Gly Asn. Phe Lys Asp Ala 610 615 62O Lieu. Glu Lieu. Lieu. Gly Ala Gly Ile Lieu. Lieu. Glu Phe Glu Pro Glu Lieu. 625 630 635 64 O Lieu. Ile Pro Thr Ile Leu Val Phe Thr Ile Llys Ser Phe Leu Gly Ser 645 650 655 US 2009/01171.57 A1 May 7, 2009 87
- Continued Ser Asp Asn Lys Asn Llys Val Ile Lys Ala Ile Asn. Asn Ala Lieu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Llys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 68O 685 Trp Met Thr Lys Ile Asn Thr Glin Phe Asn Lys Arg Lys Glu Gln Met 690 695 7 OO Tyr Glin Ala Lieu. Glin Asn Glin Val Asn Ala Lieu Lys Ala Ile Ile Glu 7 Os 71O 71s 72O Ser Lys Tyr Asn. Ser Tyr Thr Lieu. Glu Glu Lys Asn. Glu Lieu. Thir Asn 72 73 O 73 Llys Tyr Asp Ile Glu Glin Ile Glu Asn. Glu Lieu. Asn Gln Llys Val Ser 740 74. 75O Ile Ala Met Asn. Asn. Ile Asp Arg Phe Lieu. Thr Glu Ser Ser Ile Ser 75s 760 765 Tyr Lieu Met Lys Lieu. Ile Asn. Glu Val Lys Ile Asn Llys Lieu. Arg Glu 770 775 78O Tyr Asp Glu Asn. Wall Lys Thr Tyr Lieu. Lieu. Asp Tyr Ile Ile Llys His 78s 79 O 79. 8OO Gly Ser Ile Lieu. Gly Glu Ser Glin Glin Glu Lieu. Asn. Ser Met Val Ile 805 810 815 Asp Thr Lieu. Asn Asn Ser Ile Pro Phe Llys Leu Ser Ser Tyr Thr Asp 82O 825 830 Asp Llys Ile Lieu. Ile Ser Tyr Phe Asn Llys Phe Phe Lys Arg Ile Llys 835 84 O 845 Ser Ser Ser Val Lieu. Asn Met Arg Tyr Lys Asn Asp Llys Tyr Val Asp 850 855 860 Thir Ser Gly Tyr Asp Ser Asn. Ile Asn. Ile Asin Gly Asp Val Tyr Lys 865 87O 87s 88O Tyr Pro Thr Asn Lys Asn Glin Phe Gly Ile Tyr Asn Asp Llys Lieu. Ser 885 890 895 Glu Val Asn. Ile Ser Glin Asn Asp Tyr Ile Ile Tyr Asp Asn Llys Tyr 9 OO 9 OS 910 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 92O 925 Lys Ile Val Asn. Wall Asn. Asn. Glu Tyr Thir Ile Ile Asn. Cys Met Arg 930 935 94 O Asp Asn. Asn. Ser Gly Trp Llys Val Ser Lieu. Asn His Asn. Glu Ile Ile 945 950 955 96.O Trp. Thir Lieu. Glin Asp Asn. Ser Gly Ile Asin Gln Llys Lieu Ala Phe Asn 965 97O 97. Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Llys Trp Ile Phe 98O 985 990 Val Thir Ile Thr Asn Asp Arg Lieu. Gly Asp Ser Llys Lieu. Tyr Ile Asn 995 1 OOO OOS Gly Asn Lieu. Ile Asp Llys Llys Ser Ile Lieu. Asn Lieu. Gly Asn. Ile His 1010 1 O15 O2O Val Ser Asp Asn. Ile Lieu. Phe Lys Ile Val Asn. Cys Ser Tyr Thr Arg 1025 103 O O35 104 O Tyr Ile Gly Ile Arg Tyr Phe Asn. Ile Phe Asp Llys Glu Lieu. Asp Glu 1045 1 OSO O55 Thr Glu Ile Glin Thr Lieu. Tyr Asn. Asn. Glu Pro Asn Ala Asn. Ile Lieu. US 2009/01171.57 A1 May 7, 2009 88
- Continued
O60 O65 Of O
Lys Asp Phe Trp Luell Luell Tyr Asp Llys Glu Tyr Lieu. O7s O85
Wall Luell Asn Phe Ile Arg Thir Asp Ser
Ser Ile Arg Ser Thir Lell Ala Asn Arg 112O
Ser Gly Ile Ile Asn Asn Ser Ser
Asp Asn Asn Asp Ile Asn. Phe
Ser Thir Luell Pro Luell Asp Thir Ala Thr
Glu Ile Ser Gly Asn Arg Phe
Wall Wall Wall Ser Wall Thir Met Asn. Phe 12 OO
Asn Asn Ile Gly Luell Lieu. Gly Phe Ala Asp 215
h r W a. l Wall Ala Ser Thir His Met Arg Asp Asn. Thir 23 O
Asn Gly Phe Asn Phe Ile Ser Glu Glu His Gly Trp 245
<210 SEQ ID NO 11 <211 LENGTH: 25 &212> TYPE : PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . ... (25) <223> OTHER INFORMATION: BoNT/A di-chain loop region <4 OO SEQUENCE: 11 Cys Val Arg Gly Ile Ile Thir Ser Lys Thir Lys Ser Lieu. Asp Llys Gly 1. 5 15 Tyr Asn Lys Ala Lieu. Asn Asp Lieu. Cys 25
<210 SEQ ID NO 12 <211 LENGTH: &212> TYPE : PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (10) <223> OTHER INFORMATION: BoNT/B di-chain loop region
<4 OO SEQUENCE: 12 Cys Llys Ser Wall Lys Ala Pro Gly Ile Cys 1. 5
<210 SEQ ID NO 13 <211 LENGTH: 17 &212> TYPE : PRT <213> ORGANISM: Artificial Sequence US 2009/01171.57 A1 May 7, 2009 89
- Continued
&220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (17) <223> OTHER INFORMATION: BoNT/C1 di- chain loop region <4 OO SEQUENCE: 13 Cys His Lys Ala Ile Asp Gly Arg Ser Lieu. Tyr Asn Llys Thir Lieu. Asp 1. 5 1O 15 Cys
<210 SEQ ID NO 14 <211 LENGTH: 14 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (14) <223> OTHER INFORMATION: BoNT/D di-chain loop region <4 OO SEQUENCE: 14 Cys Lieu. Arg Lieu. Thir Lys Asn. Ser Arg Asp Asp Ser Thr Cys 1. 5 1O
<210 SEQ ID NO 15 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (15) <223> OTHER INFORMATION: BoNT/E di-chain loop region <4 OO SEQUENCE: 15 Cys Lys Asn. Ile Val Ser Wall Lys Gly Ile Arg Llys Ser Ile Cys 1. 5 1O 15
<210 SEQ ID NO 16 <211 LENGTH: 17 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (17) <223> OTHER INFORMATION: BoNT/F di-chain loop region <4 OO SEQUENCE: 16 Cys Llys Ser Val Ile Pro Arg Lys Gly. Thir Lys Ala Pro Pro Arg Lieu. 1. 5 1O 15 Cys
<210 SEQ ID NO 17 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (15) <223> OTHER INFORMATION: BoNT/G di-chain loop region <4 OO SEQUENCE: 17 Cys Llys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu Glin Cys 1. 5 1O 15
<210 SEQ ID NO 18 US 2009/01171.57 A1 May 7, 2009 90
- Continued
<211 LENGTH: 29 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (29) <223> OTHER INFORMATION: TeNT di-chain loop region <4 OO SEQUENCE: 18 Cys Llys Lys Ile Ile Pro Pro Thr Asn. Ile Arg Glu Asn Lieu. Tyr Asn 1. 5 1O 15 Arg Thr Ala Ser Lieu. Thir Asp Lieu. Gly Gly Glu Lieu. Cys 2O 25
<210 SEQ ID NO 19 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (15) <223> OTHER INFORMATION: BaNT di-chain loop region <4 OO SEQUENCE: 19 Cys Llys Ser Ile Val Ser Llys Lys Gly. Thir Lys Asn. Ser Lieu. Cys 1. 5 1O 15
<210 SEQ ID NO 2 O <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: DOMAIN <222> LOCATION: (1) . . . (15) <223> OTHER INFORMATION: BuNT di-chain loop region <4 OO SEQUENCE: 2O Cys Lys Asn. Ile Val Ser Wall Lys Gly Ile Arg Llys Ser Ile Cys 1. 5 1O 15
<210 SEQ ID NO 21 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (5) <223> OTHER INFORMATION: Bovine enterokinase protease cleavage site. <4 OO SEQUENCE: 21 Asp Asp Asp Asp Llys 1. 5
<210 SEQ ID NO 22 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (1) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site consensus sequence <221 NAME/KEY: VARIANT <222> LOCATION: (2) ... (3) <223> OTHER INFORMATION: Xaa can be amino amino acid <221 NAME/KEY: VARIANT <222> LOCATION: (5) . . . (5) US 2009/01171.57 A1 May 7, 2009 91
- Continued
<223> OTHER INFORMATION: Xaa can be amino amino acid
<4 OO SEQUENCE: 22 Glu Xaa Xala Tyr Xaa Glin Gly 1. 5
<210 SEQ ID NO 23 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site consensus sequence <221 NAME/KEY: VARIANT <222> LOCATION: (2) ... (3) <223> OTHER INFORMATION: Xaa can be any amino acid <221 NAME/KEY: VARIANT <222> LOCATION: (5) . . . (5) <223> OTHER INFORMATION: Xaa can be any amino acid <4 OO SEQUENCE: 23 Glu Xaa Xala Tyr Xaa Glin Ser 1. 5
<210 SEQ ID NO 24 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site.
<4 OO SEQUENCE: 24 Glu Asn Lieu. Tyr Phe Glin Gly 1. 5
<210 SEQ ID NO 25 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site.
<4 OO SEQUENCE: 25 Glu Asn Lieu. Tyr Phe Glin Ser 1. 5
<210 SEQ ID NO 26 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site.
<4 OO SEQUENCE: 26 Glu Asin Ile Tyr Thr Glin Gly 1. 5
<210 SEQ ID NO 27 US 2009/01171.57 A1 May 7, 2009 92
- Continued
<211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site.
<4 OO SEQUENCE: 27 Glu Asin Ile Tyr Thr Glin Ser 1. 5
<210 SEQ ID NO 28 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site.
<4 OO SEQUENCE: 28 Glu Asn. Ile Tyr Lieu. Glin Gly 1. 5
<210 SEQ ID NO 29 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site.
<4 OO SEQUENCE: 29 Glu Asn. Ile Tyr Lieu. Glin Ser 1. 5
<210 SEQ ID NO 3 O <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site. <4 OO SEQUENCE: 30 Glu Asn Val Tyr Phe Glin Gly 1. 5
<210 SEQ ID NO 31 <211 LENGTH: 7 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <221 NAME/KEY: SITE <222> LOCATION: (1) . . . (7) <223> OTHER INFORMATION: Tobacco Etch Virus protease cleavage site. <4 OO SEQUENCE: 31 Glu Asn Val Tyr Ser Glin Ser 1. 5
<210 SEQ ID NO 32 <211 LENGTH: 7