CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser. No.

62/412,456, filed October 25, 2016, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number ROl A095274 awarded by National Institute of Allergy and Infectious Diseases. The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure relates to neurotoxins and uses thereof. In particular, provided herein are botulinum neurotoxins with altered properties and uses thereof (e.g., research, screening, and therapeutic uses).

BACKGROUND OF THE DISCLOSURE

Botulinum neurotoxins (BoNTs), synthesized by the Gram-positive, soil-dwelling bacterium Clostridium botulinum, are the most toxic substances known to humankind and are the causative agents of the neuroparalytic disease botulism (Johnson E (2005) in Topley and Wilson's microbiology and microbial infections, ed S. P. Bordello, P. R. Murray,and G.Funke (Hodder Arnold, London, United Kingdom), pp 1035-1088). Seven immunologically distinct serotypes of BoNTs designated A through G have been described (Gimenez DF & Gimenez JA (1995) Int J Food Microbiol 27: 1-9). BoNTs are initially synthesized as a single-chain polypeptide of ~150kDa, but posttranslational proteolytic cleavage yields distinct heavy and light chains (HC and LC) of -lOOkDa and ~50kDa linked by a disulfide bond. The HC is further functionally divided into the HCc and HC _N sub-domains. The HCc domain is responsible for recognition and binding to specific neuronal cell surface receptors leading to endocytosis, while the HC _N domain is responsible for channel formation in the endocytic vesicle membrane and translocation and internalization of the LC across the endosomal membrane (Montecucco et al., (2004) Trends Microbiol 12: 442-446; Fischer A & Montal M (2007) J Biol Chem 282: 29604-29611; Fischer A, et al (2009) Proc Natl Acad Sci USA 106: 1330-1335). After translocation, the disulfide bond is cleaved, and the LC is released into the cell cytosol and refolded to the active enzyme component as a zinc-dependent endopeptidase (Fischer et al., supra; Fischer A & Montal M (2007) Proc Natl Acad Sci U SA 104: 10447-10452; Pirazzini, et al., Cell Rep 2014, 8, 1870-1878). The LC then specifically targets and cleaves an intracellular SNARE protein at the pre-synaptic vesicles, which leads to inhibition of neurotransmitter release. Each BoNT serotype has a distinct cleavage target, with BoNT/A and E cleaving SNAP-25 at distinct sites, BoNT/B, D, F, and G cleaving VAMP/synaptobrevin at different sites, and BoNT/C cleaving both SNAP-25 and syntaxin (reviewed in Montecucco C & Schiavo G (1994) Mol Microbiol 13 : 1-8).

BoNT/A and to a much lesser extent BoNT/B are used as unique and important pharmaceuticals to treat a variety of neuromuscular disorders and in cosmetics. Conditions for which the Food and Drug Administration approved the use of BoNTs include cosmetic treatments and to temporarily relieve a variety of muscle spasticity disorders, hyperhydrosis and migraines (Chaddock JA & Acharya KR (2011) FEBS J 27%: 899-904). Cosmetic and clinical applications of BoNTs are increasing, and new formulations of BoNTs for pharmaceutical purposes are being developed necessitating clinical trials, accurate potency determination, and neutralizing antibody screening. For example, BoNTs are

pharmaceutically administered for the treatment of pain disorders, voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in lubag, dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in

Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non- kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior

hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, and myelon tumor.

BoNT with optimized properties for the particular condition being treated or other usage are needed.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to neurotoxins and uses thereof. In particular, provided herein are botulinum neurotoxins with altered properties and uses thereof (e.g., research, screening, and therapeutic uses).

Provided herein are BoNT/FA toxins and uses thereof (e.g., therapeutic uses). In some embodiments, the BoNT/FA is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: l . In some embodiments, the heavy or light chain of the BoNT/FA is combined in a chimeric BoNT with a heavy or light chain from a different serotype of BoNT. Further embodiments provide nucleic acids and vectors encoding such amino acid sequences.

The BoNT/FA toxins described herein find use in a variety of therapeutic and cosmetic applications (e.g., pain disorders, voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in lubag, dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non-kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, or myelon tumor). Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows potency of BoNT toxins in cultured human neurons.

FIG.2 shows a) duration of action of BoNT/FA, BoNT/Fl, and BoNT/Bl in cultured hiPSC derived neurons; and b) and c) local and systemic effects in mice after intramuscular injection of BoNT/Bl or BoNT/FA .

FIG. 3 shows the amino acid sequence of SEQ ID NO: 1 (BoNT/FA).

DEFINITIONS

To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:

As used herein, the term "exhibits one or more altered properties" (e.g., altered duration of action relative to wild type BoNT, altered neuronal selectivity relative to wild type BoNT, altered potency relative to wild type BoNT, altered onset of action relative to wild type BoNT, or altered uptake or transport relative to wild type BoNT) refers to a property of a variant or hybrid BoNT (e.g., those described herein) that is altered relative to a wild type BoNT.

As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cell, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.

As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos and stem cell derived cells. As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions {e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. {See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975]).

As used herein, the term "purified" or "to purify" refers to the removal of components

{e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non- immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include cells, tissues, blood products, such as plasma, serum and the like. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to hybrid neurotoxins and uses thereof. In particular, provided herein are chimeric botulinum neurotoxins with altered properties and uses thereof (e.g., research, screening, and therapeutic uses).

Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of an eighth serotype of BoNT, designated "type H," has stirred controversy. This BoNT was produced together with BoNT/B2 in a dual -toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and the novel toxin and from sequence information. However, data from two independent laboratories indicated neutralization by antibodies raised against BoNT/Al, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/Al receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/Fl .

Described herein is characterization of this toxin (FIG. 3; SEQ ID NO: l) purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 xlO ⁷ mouse 50% lethal dose

(mLD ₅ o)/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC ₅₀ ] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/Al, although at 16- to 20-fold-lower efficiency than for BoNT/Al .

Further analyses of the purified BoNT/FA have revealed that, surprisingly, the half- life in cultured human neurons is similar to that of BoNT/Bl (currently marketed as

Myobloc) (FIG. 1). This was unexpected because the light chain of this toxin is similar to BoNT/F, and BoNT/F serotypes are known to have a very short duration of action. In addition, local injection into mice resulted in a similar maximum local paralysis response and similar duration as BoNT/Bl, however, systemic symptoms were milder at the same concentrations, had a later onset and slightly slower recovery (Fig.2). This indicates that this toxin provides an alternative to BoNT/Bl as a local paralytic therapeutic and may have a larger safety margin. As with BoNT/Bl, the therapeutic target is VAMP1 and 2, but this toxin enters cultured human neuronal cells about 10,000 fold more efficiently and has an at least similar duration of action as BoNT/Bl . In addition, derivatives of BoNT/FA or engineered hybrid toxins maintaining specific structural domains of BoNT/FA yield therapeutics with advantageous and novel characteristics, such as faster cell entry and unique in vivo distribution.

This BoNT variant is over 10 fold more potent than BoNT/Al and over 1000 fold more potent than BoNT/Bl in cultured human neurons (Fig. 1). While BoNT/Al cleaves SNAP-25, BoNT/FA cleaves VAMP. BoNT/Bl also cleaves VAMP, but it does not enter human neurons as efficiently as BoNT/Al and BoNT/FA. Importantly, BoNT/FA has similar duration of action in human neurons and in mice as BoNT/Bl and caused similar local paralysis but with milder overall motorneuron deficiency.

In some embodiments, BoNT molecules are prepared using a manufacturing process described, for example, in Dressier D. HNO. 2012 Jun;60(6):496-502; Wortzman MS, Pickett A. Aesthet Surg J. 2009 Nov;29(6 Suppl):S34-42; each of which is herein incorporated by reference in its entirety or another suitable method.

In some embodiments, BoNT molecules are provided as pharmaceutical

compositions. The compounds described herein, optionally together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical formulations of sterile injectable solutions for parenteral (including subcutaneous) use. Such

pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.

BoNT compositions are typically administered via injection, although other delivery methods are specifically contemplated.

The dose, when using the compounds and formulations described herein, can vary within wide limits and as is customary and is known to the physician, it is to be tailored to the individual conditions in each individual case. It depends, for example, on the nature and severity of the illness to be treated, on the condition of the patient, on the compound employed or on whether an acute or chronic disease state is treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds. Representative doses include, but not limited to, about 0.1 pg to about 5000 ng, about 0.001 ng to about 2500 ng, about 0.001 ng to about 1000 ng, 0.001 ng to about 500 ng, 0.001 ng to about 250 ng, about 0.001 ng to 100 ng, about 0.001 ng to about 50 ng and about 0.001 ng to about 25 ng. Multiple doses may be administered during the day, especially when relatively large amounts are deemed to be needed, for example 2, 3 or 4 doses. Depending on the individual and as deemed appropriate from the patient's physician or caregiver it may be necessary to deviate upward or downward from the doses described herein.

The amount of active ingredient, for use in treatment will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will ultimately be at the discretion of the attendant physician or clinician. In general, one skilled in the art understands how to extrapolate in vivo data obtained in a model system, typically an animal model, to another, such as a human. In some circumstances, these extrapolations may merely be based on the weight of the animal model in comparison to another, such as a mammal, preferably a human, however, more often, these extrapolations are not simply based on weights, but rather incorporate a variety of factors. Representative factors include the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized, on whether an acute or chronic disease state is being treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds described herein and as part of a drug

combination. The dosage regimen for treating a disease condition with the compounds and/or compositions is selected in accordance with a variety factors as cited above. Thus, the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods described herein.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. The daily dose can be divided, especially when relatively large amounts are administered as deemed appropriate, into several, for example 2, 3 or 4 part administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the daily dose indicated.

For preparing pharmaceutical compositions, the selection of a suitable

pharmaceutically acceptable carrier is typically liquid. Liquid form preparations include solutions, suspensions and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed, as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds according may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.

In some embodiments, compositions comprising BoNT molecules comprise purified BoNT stabilized and preserved, e.g. with human serum albumin and non-protein stabilizers. These may be derived from non-human and non-animal sources. In some embodiments, BoNTs are provided in combination with native complexes. In some embodiments, compositions are in a pH neutral buffer and stabilized with human serum albumin in stabilized liquid formulation. In some embodiments, compositions are lyophilized for re- suspension in saline or sterile water.

The BoNT preparations described herein find use in the treatment of a variety of conditions including, but not limited to, cosmetic treatments and to temporarily relieve a variety of muscle spasticity disorders, hyperhydrosis and migraines (Chaddock JA & Acharya KR (201 \) FEBS J 278: 899-904; Dressier, D. Clinical applications of botulinum toxin. In Curr opin microbiol, England, 2012; Vol. 15, pp 325-336; Dressier, D. Botulinum toxin drugs: Brief history and outlook. J Neural Transm (Vienna) 2016, 123, 277-279.). Cosmetic and clinical applications of BoNTs are increasing, and new formulations of BoNTs for pharmaceutical purposes are being developed necessitating clinical trials, accurate potency determination, and neutralizing antibody screening. For example, BoNTs are

pharmaceutically administered for the treatment of pain disorders, voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in lubag, dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in

Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non- kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior

hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, and myelon tumor.

Experimental

Example 1

This Example describes properties of BoNT/B l, BoNT/F l, and BoNT/FA. HiPSC derived neurons were exposed to serial dilutions of BoNT/Bl, BoNT/Fl, or BoNT/FA for 48 h followed by removal of all extracellular toxin. Cells were incubated at 37°C, fed every 2-3 days, and harvested at the indicated time points after toxin exposure. The percentage of VAMP2 cleavage was determined by Western blot and densitometry. The EC50 values were determined by curve fit using PRISM6 software, and the half-life (tl/2) for LC activity, as measured by VAMP2 recovery, was calculated from derived EC50 values at the three time points. Results are shown in FIG. 1.

The indicated doses of BoNT/FA or BoNT/Bl were injected into the gastrocnemius muscle of ICR mice. The onset and duration of local paralysis were determined by DAS assay using a scale from 0 (no symptoms) to 5 (maximal paralysis), and overall (systemic) motor-neuron deficiency was measured by Rotarod using an accelerating cycle (4-40 rpm over 5 min) and measuring the amount of time mice remained running on the rod (latency time). Results are shown in FIG. 2. All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the disclosure will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.