阅读说明：本技术 肉毒神经毒素用于治疗震颤的新用途 (New use of botulinum neurotoxin for the treatment of tremors ) 是由 J·西科斯 I·普莱特 R·赫蒙泽 M·阿尔图斯 C·娜莱斯瑞 D·辛普森 B·贾巴里 于 2020-02-18 设计创作，主要内容包括：本发明涉及肉毒神经毒素在治疗震颤中的新用途,特别地涉及供治疗上肢震颤使用的肉毒神经毒素,所述治疗上肢震颤包括将肉毒神经毒素施用于前臂/腕部、肘部和肩部的至少一块肌肉,其中将肉毒神经毒素以2至6U范围的剂量施用于前臂/腕部的选自尺侧腕伸肌(ECU)、桡侧腕伸肌(ECR)、旋前方肌(PQ)和旋后肌的组的至少一块肌肉,并且其中将肉毒神经毒素以约20U的剂量施用于肘部的至少一块肌肉,并以约15U的剂量施用于肩部的至少一块肌肉。(The present invention relates to a novel use of a botulinum neurotoxin in the treatment of tremors, in particular to a botulinum neurotoxin for use in the treatment of upper limb tremors comprising applying a botulinum neurotoxin to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is applied to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle in a dose ranging from 2 to 6U, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow in a dose of about 20U and to at least one muscle of the shoulder in a dose of about 15U.)

1. A botulinum neurotoxin for use in the treatment of upper limb tremor comprising applying the botulinum neurotoxin to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is applied to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle in a dose ranging from 2 to 6U, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow in a dose of about 20U and to at least one muscle of the shoulder in a dose of about 15U.

2. The botulinum neurotoxin for use according to claim 1, wherein the botulinum neurotoxin is additionally administered to at least one muscle of the wrist/forearm selected from the group of the radial wrist Flexor (FCR), the ulnar wrist Flexor (FCU) and the circumflex round muscle (PT) in a dose ranging from 4 to 16U per muscle.

3. The botulinum neurotoxin for use according to claim 1, wherein the botulinum neurotoxin is administered at a dose of about 2.5U to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator.

4. The botulinum neurotoxin for use according to claim 3, wherein the botulinum neurotoxin is additionally administered to at least one muscle selected from the group of the radial wrist Flexor (FCR), the ulnar wrist Flexor (FCU) and the circumflex (PT) of the wrist/forearm at a dose of about 10U per muscle.

5. The botulinum neurotoxin for use according to any one of claims 1 to 4, wherein the botulinum neurotoxin is not applied to extensor digitorum communis.

6. The botulinum neurotoxin for use according to any one of claims 1 to 5, wherein the total dose of botulinum neurotoxin applied to the forearm/carpus muscle does not exceed 65U.

7. The botulinum neurotoxin for use according to any one of claims 1 to 6, wherein the botulinum neurotoxin is applied to at least one muscle of the elbow selected from the group of the brachial and the triceps brachii muscles.

8. The botulinum neurotoxin for use according to any one of claims 1 to 7, wherein the botulinum neurotoxin is not applied to the biceps brachii muscle.

9. The botulinum neurotoxin for use according to any one of claims 1 to 8, wherein the total dose of botulinum neurotoxin applied to an elbow muscle does not exceed 40U.

10. The botulinum neurotoxin for use according to any one of claims 1 to 9, wherein the botulinum neurotoxin is applied to at least one muscle of the shoulder selected from the group of latissimus dorsi, pectoralis major, supraspinatus and infraspinatus.

11. The botulinum neurotoxin for use according to any one of claims 1 to 10, wherein the botulinum neurotoxin is not applied to the deltoid and the great muscle.

12. The botulinum neurotoxin for use according to any one of claims 1 to 11, wherein the total dose of botulinum neurotoxin applied to a shoulder muscle does not exceed 60U.

13. The botulinum neurotoxin for use according to any one of claims 1 to 12, wherein the total dose of botulinum neurotoxin applied to the forearm/wrist, elbow and shoulder muscles does not exceed 165U.

14. The botulinum neurotoxin for use according to claim 1, wherein the botulinum neurotoxin is administered to 4, 5, 6 or 7 muscles of the forearm/wrist, 2 muscles of the elbow and 4 muscles of the shoulder at a dosage according to the following protocol:

15. the botulinum neurotoxin for use according to claim 1, wherein the botulinum neurotoxin is administered to 7 muscles of the forearm/wrist, 2 muscles of the elbow and 4 muscles of the shoulder at a dosage according to the following protocol:

16. the botulinum neurotoxin for use according to any one of claims 1 to 15, wherein the botulinum neurotoxin is a neurotoxic component of a botulinum neurotoxin complex, wherein the neurotoxic component is devoid of any other protein component of a botulinum neurotoxin complex.

17. The botulinum neurotoxin for use according to any one of claims 1 to 16, wherein the botulinum neurotoxin is selected from the group of serotypes consisting of A, B and E.

18. The botulinum neurotoxin for use according to any one of claims 1 to 17, wherein the botulinum neurotoxin is administered together with at least one standard therapeutic agent selected from propranolol, paminone, any other antiepileptic drug or calcium channel blocker, Deep Brain Stimulation (DBS), magnetic resonance guided high frequency ultrasound (MRgHiFUS), local electrical stimulation, biofeedback, kinematic assessment guided stimulation, anti-tremor device, anti-tremor smartphone application or a combination thereof.

19. A pharmaceutical composition comprising the botulinum neurotoxin according to any one of claims 1 to 18 for use in the treatment of upper limb tremors.

20. A method of treating upper limb tremor, wherein the method comprises administering a therapeutically effective amount of the botulinum neurotoxin of any one of claims 1 to 19.

Technical Field

The present invention relates to a novel use of a botulinum neurotoxin in the treatment of tremors, in particular the use of a botulinum neurotoxin in the treatment of upper limb tremors of adults or children suffering from essential tremors or for any other reason in which a reduction of tremors provides a benefit to a subject.

Background

The clostridium genus is a genus of anaerobic gram-positive bacteria belonging to the phylum firmicutes. The genus clostridium consists of approximately 100 species, including common free-living bacteria as well as important pathogens, such as clostridium botulinum and clostridium tetani. These two species produce neurotoxin, botulinum toxin and tetanus toxin, respectively. These neurotoxins are potent inhibitors of calcium-dependent neurotransmitter secretion by neuronal cells and are the strongest toxins known to humans. The human lethal dose is between 0.1ng and 1ng per kilogram of body weight.

Oral ingestion of botulinum toxin via contaminated food or production of botulinum toxin in wounds can cause botulism, which is characterized by paralysis of various muscles. Paralysis of the respiratory muscles can lead to death in the affected individual.

While both botulinum neurotoxin (BoNT) and tetanus neurotoxin (TxNT) act through similar initial physiological mechanisms of action, inhibiting neurotransmitter release from axons of affected neurons into synapses, their clinical responses differ. While botulinum toxin acts on neuromuscular junctions and other cholinergic synapses in the peripheral nervous system, it inhibits the release of the neurotransmitter acetylcholine, thereby causing flaccid paralysis; tetanus toxin, which is transcytosed into central neurons, acts primarily in the central nervous system, preventing the release of the inhibitory neurotransmitters GABA (gamma-aminobutyric acid) and glycine by degrading proteins (synaptophysin). Subsequent hyperactivation of spinal motoneurons causes a generalized contraction of the agonistic and antagonistic muscle groups, which is called tonic spasm (hard paralysis).

While tetanus neurotoxin exists in one immunologically distinct type, botulinum neurotoxin is known to exist in seven distinct immunogenic serotypes, referred to as BoNT/A through BoNT/G, and having further subtypes. Most strains of clostridium botulinum produce one type of neurotoxin, but strains that produce multiple toxins are also described.

Botulinum neurotoxin and tetanus neurotoxin have highly homologous amino acid sequences and exhibit similar domain structures. Their biologically active form comprises two peptide chains linked by disulfide bonds: an about 50kDa light chain and an about 100kDa heavy chain. The linker or loop region, which varies in length from clostridial toxin, is located between the two cysteine residues that form the disulfide bond. This loop region is subjected to proteolytic cleavage by an unknown clostridial endoprotease to obtain the biologically active toxin.

The molecular mechanisms of poisoning by TxNT and BoNT also appear to be similar: entry into a target neuron mediated by the binding of the C-terminal portion of the heavy chain to a specific cell surface receptor; the toxin is then absorbed by receptor-mediated endocytosis. The low pH in the endosome so formed then triggers a conformational change in the clostridial toxin which enables the toxin to intercalate itself into the endosomal membrane and translocate through the endosomal membrane into the cytoplasm, where the disulfide bond joining the heavy and light chains is reduced. The light chain can then selectively cleave so-called SNARE proteins that are essential for the different steps of neurotransmitter release into the synaptic cleft (e.g. recognition, docking and fusion of neurotransmitter-containing vesicles with the plasma membrane). TxNT, BoNT/B, BoNT/D, BoNT/F and BoNT/G cause proteolytic cleavage of synaptobrevin or VAMP (vesicle-associated membrane protein), BoNT/A and BoNT/E cleave plasma membrane-associated protein SNAP-25, and BoNT/C cleaves intact plasma membrane protein synaptotagmin and SNAP-25.

In clostridium botulinum, the botulinum toxin is formed as a protein complex comprising a neurotoxic component and a non-toxic protein. The accessory protein embeds the neurotoxic component, thereby protecting it from degradation by digestive enzymes in the gastrointestinal tract. Thus, most serotypes of botulinum neurotoxin are orally toxic. Complexes with, for example, 450kDa or 900kDa can be obtained from cultures of Clostridium botulinum.

In recent years, botulinum neurotoxins have been used, for example, as therapeutic agents for the treatment of dystonia and spasticity, and have also been used in cosmetic applications, such as the treatment of fine lines. The preparation comprising botulinum toxin complexes may be for example obtained from Ipsen LtdOr Allergan Inc.Are commercially available. Highly pure neurotoxic components free of any complexing proteins are available, for example, from Merz Pharmaceuticals GmbH of Frankfurt

Clostridial neurotoxins are typically injected into the affected muscle tissue, bringing the agent close to the neuromuscular end plate, i.e., close to the cellular receptor that mediates uptake of the agent into the nerve cells controlling the affected muscle. Different degrees of neurotoxin spread have been observed. Neurotoxin spread is believed to depend on the amount injected and the particular neurotoxin preparation. It may lead to adverse side effects such as paralysis of nearby muscle tissue, which can be largely avoided by reducing the injected dose to therapeutically relevant levels. Overdosing may also trigger the immune system to produce neutralizing antibodies that inactivate the neurotoxin, thereby preventing the neurotoxin from alleviating involuntary muscle activity. Immunotolerance to botulinum toxin has been shown to correlate with cumulative doses.

Clostridial neurotoxins exhibit variable serotype-specific duration of action. The clinical therapeutic effect of BoNT/a persists for approximately 3 months for neuromuscular disorders and 6 to 12 months for hyperhidrosis. On the other hand, BoNT/E action lasts for about 4 weeks. One possible explanation for the different duration of action may be the different subcellular localization of the BoNT serotypes. The protease domain of the BoNT/A light chain is localized in a punctate manner to the plasma membrane of neuronal cells, co-localized with its substrate SNAP-25. In contrast, the short-lasting BoNT/E serotypes are cytoplasmic. Membrane association may protect BoNT/A from cytosolic degradation mechanisms, thereby prolonging BoNT/A survival in neuronal cells.

The longer lasting therapeutic effect of BoNT/a compared to other serotypes (e.g., B, C, D, E, F, G serotype) makes it more suitable for certain clinical uses, particularly certain cosmetic uses.

Tremor is the most common movement disorder. Its unmet medical need is manifested in a variety of clinical conditions that result in tremor symptoms in different body parts. These symptoms include, for example, jerky, rhythmic movements of the head, arms, hands, fingers, legs, feet, torso, vocal cords, or involuntary additional movements thereof. As a result, dysfunction of the affected body part and a reduction in the quality of life of the subject occur. By definition, tremor is an oscillating involuntary movement of muscles that can occur at rest or activity. During voluntary movements (action tremor), the tremor may affect the posture of a body part, depending on the movement. The corresponding underlying disease and tremor symptoms and syndromes are not clearly distinguished clinically phenomenologically. The consensus on tremor syndrome was recently elucidated (Bhatia et al, motion Disorders, vol 33, stage 1, 2018), but it is suggested that consistent use of the term has not been achieved. Due to the unclear etiology in some cases (e.g., essential tremor) and the pathological mechanisms of the underlying disorder, it is expected that modifications to this consensus will be needed in the future.

Tremor is of diverse etiology and may be associated with physiological function (cold-induced physiological tremor), pathological origin (dyskinetic tremor), parkinson's tremor, helminth tremor, essential tremor, drug-related side effects, alcohol or drug withdrawal symptoms (tremor delirium), or functional tremor (also known as psychogenic tremor). The coexistence of these factors renders it challenging to clearly differentiate the etiologies. Descriptors like diagnosis or symptoms as listed above are used according to the international consensus statement of Parkinson's disease and the society of dyskinesias (IPMDS) tremor special working group's clinical diagnostic criteria [ Bhatia et al, Movement Disorders, Vol.33, phase 1, 2018 ] and earlier.

Essential Tremor (ET) is the most common adult dyskinesia [ Hess ET al, Tremor Other hyperkinetic Mov 2012; 2 ], [ Jankovic et al, motion Disorders, Vol.11, No. 3, 1996, p.250-. ET diagnosis is encoded in ICD-10(2018) with G250(G25.0), G250(G25.0) uniquely describing this medical entity. A recent prevalence estimate [ Louis ET al, Tremor and Other Hyperkinetic Movements 2014] calculated that approximately 700 million American citizens have ET (including hand, head and palate expressions). Existing therapies have several drawbacks and do not adequately treat the entire patient population. Oral drugs (the FDA's only approved drug propranolol for ET, and promethione for off-label use) have been used since 1970, but their response rates have been inadequate. In addition, these treatments are associated with a high rate of systemic adverse events (e.g., hypotension, a sedated state, nausea, ataxia, or confusion). The pathogenic role of beta blockers like propranolol in the development of Parkinson's disease has recently been arguably discussed [ Mittal et al, Science 357,891-898(2017), and thus represents a questionable approach to treatment. Deep Brain Stimulation (DBS) is a powerful but invasive treatment option, and MRI-guided high-frequency ultrasound thalamotomy (mrghhifu) is effective, but it induces permanent lesions in the brain. Both can only be offered to patients with severe disabling tremor at the highly specialized neurosurgical center.

Treatment options for tremor generally focus on conservative measures (pharmacotherapy), but the involuntary mechanism of tremor is difficult to treat adequately in patients with progressive neurological diseases such as parkinson's disease. Thus, treatment of tremor has focused on reducing the amplitude of the oscillations. The earliest routes used ethanol (a well-known recreational substance that inhibits glutamatergic neuroleptic activity). The antihypertensive and antiarrhythmic drug propranolol (beta adrenergic blocker) was later used as an anti-tremor drug, etc. Several derivatives of beta-adrenergic blockers from the antihypertensive drug group have been tested and used off-label in this indication.

Neurosurgical treatment options provide a destructive solution to the brain center of the thalamus known to be involved in the tremor circuit, i.e. transthalamotomy destruction, with magnetic resonance guided high frequency ultrasound therapy being the current solution. Deep brain stimulation of specific brain regions has also been reported to be effective in treating tremor disorders in a limited number of patients.

Another therapeutic alternative is botulinum toxin a, which is used in a wide variety of diseases and medical conditions, including cervical dystonia, spasticity, incontinence, migraine, and the like. Botulinum toxin a is also used to treat, for example, dystonic tremor, task-specific tremor, parkinson tremor and essential tremor. Intramuscular injection of botulinum toxin A relieves muscle tone in the treated muscle, thereby reducing tremor amplitude by causing a localized partial weakness in the muscles involved.

Topical treatment of tremor by applying a botulinum neurotoxin injection to the tremor muscle has been challenging, as tremor can affect the entire arm or only a portion of it, causing a wide range of clinical phenomena, depending on the underlying disease and tremor pattern. Treatment of upper limb tremor by administration of botulinum neurotoxin to several muscles of the carpus and forearm (primarily into the wrist flexors and extensors) in a fixed dose/fixed muscle route has shown strong efficacy, but also debilitating side effects. Adverse events were mainly weakness of the arms, wrists and drop fingers. These adverse events can be partially reduced by lowering the dose in subsequent treatments, by modifying the choice of muscle injected, or by switching the strategy to a low initial starting dose, followed by a slow increase in titration or booster injection. On the other hand, these changes can lead to reduced efficacy. Any low initial starting dose requires early follow-up (booster injections) to offset the initial under-treatment for tremor. Modifications of injected muscle selection intended to alter the weakness pattern of the limb may cause further weakness in other muscles, making the patient's treatment more difficult to tolerate.

Depending on the range of motion during tremor, it is also difficult to select a muscle, since ideally this should be decided based on an accurate analysis of the muscle function and its sum. Visual clinical assessment of motion direction and oscillation amplitude is inaccurate and limited by the examiner's poor discriminative power for the fuzzy components of complex motion. However, without the more accurate and reliable alternatives available, visual assessment is still routinely used to distribute muscles and dose, focusing attention on the apparent motion of the wrist (most commonly flexion and extension). Thus, the earlier route used a fixed dose for a pre-selected group of forearm muscles (primarily the wrist flexors and extensors). In earlier studies, this may have resulted in an unexpectedly high frequency of wrist muscle weakness. However, tremor movements of the arm may be the result of tremors of additional muscles of the arm (elbow and shoulder muscles). The range of motion of these joints adds to the tremor of the wrist, resulting in a categorical (subsuming) overall effect. In addition, other parts of the body (e.g., lower limbs, head, trunk) may also have tremors, which may cause instability in the case of oscillatory waves transmitted to remote body parts such as the wrist. Without the typical oscillation pattern, in upper limb tremor of the hand, tremor motion can affect both the proximal and distal muscle groups (wrist and shoulder motor muscles) in parallel, without the typical oscillation pattern being produced. It is difficult for a clinician to distinguish visually detected individual components of a classified tremor (tremor of individual muscles or even muscle groups) by visual inspection alone.

There are technical measures available for differentiating the contribution of muscle groups to tremor, such as kinematic measurements or electromyographic EMG. However, these techniques are not widely spread in clinical practice for analyzing tremor. In particular, needle EMG is not conducive to the examination of a large number of muscles, as this is a painful procedure. A further challenge is that no training has been established for the injector, nor have generally accepted guidelines for muscle and dose selection strategies for treating tremors with botulinum toxin a. The selection of the appropriate muscles and the correct dosage for each muscle is critical to the efficacy and safety of treatment for upper limb, lower limb, head and neck and vocal cord tremor. There is currently no defined total therapeutic dose window or therapeutic dose window per muscle that allows optimal treatment with adequate efficacy and tolerability. In particular, because of the general mode of action of botulinum neurotoxin, i.e. muscle paralysis, it is necessary to take into account the possible weakness of the muscle treated by the botulinum neurotoxin.

In WO2015039244, a technique for treating tremor is proposed that uses an adaptive, conventional system to determine muscle activity by upper limb kinematic analysis (primarily tremor amplitude) and calculate individual doses to be administered to each muscle. Jog et al used botulinum neurotoxin AThe applicability of this flexible dose determination was demonstrated in 19 patients in a clinical trial of (Jog et al, Poster Presented at TOXINS 2017, Madrid, Spain, 2017, month 1, 18-21).

There is a strong need for further improvements in the treatment options available for treating tremor. In particular, it is desirable to effectively treat upper limb tremor in patients suffering from essential tremor, parkinson's disease and any other upper limb tremor, and without adverse events.

Object of the Invention

It is an object of the present invention to improve the treatment of tremor, in particular to provide treatment of any tremor symptoms of the upper limbs. Furthermore, it is an object of the present invention to provide a safe and effective treatment regimen for treating upper limb tremors that is easy to administer to patients without the need for detailed tremor analysis and comprehensive tremor resolution. It is a particular object of the present invention to develop a drug delivery system for the administration of botulinum neurotoxin for use in the treatment of upper limb tremors, said system allowing an adaptive tailored administration to the muscles of the wrist and forearm in combination with an easy to administer fixed administration to the muscles of the elbow and shoulder.

Disclosure of Invention

Surprisingly, it has been identified that a botulinum neurotoxin can be advantageously used for providing a treatment of upper limb tremors if the botulinum neurotoxin is applied to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is applied to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscles at a dose ranging from 2 to 6U, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

Thus, in one aspect, the present invention relates to a botulinum neurotoxin for use in the treatment of upper limb tremor comprising applying the botulinum neurotoxin to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is applied to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle at a dose ranging from 2 to 6U, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In another aspect, the present invention relates to a pharmaceutical composition comprising a botulinum neurotoxin for use in the treatment of upper limb tremor comprising applying the botulinum neurotoxin to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is applied to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle in a dose ranging from 2 to 6U, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow in a dose of about 20U and to at least one muscle of the shoulder in a dose of about 15U.

In yet another aspect, the present invention relates to a method of treating a disease or condition associated with upper limb tremor, the method comprising administering a botulinum neurotoxin to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle in a dose ranging from 2 to 6U, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow in a dose of about 20U and to at least one muscle of the shoulder in a dose of about 15U.

Detailed Description

The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

In one aspect, the present invention relates to a botulinum neurotoxin for use in the treatment of upper limb tremor comprising applying the botulinum neurotoxin to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is applied to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle at a dose ranging from about 2 to 6U, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In a preferred embodiment of the present invention, a botulinum neurotoxin for use in the treatment of upper limb tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle at a dose ranging from about 2.5 to 5U, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In a further preferred embodiment of the present invention, a botulinum neurotoxin for use in the treatment of upper limb tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator muscle at a dose of about 2.5U, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In one embodiment of the present invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator at a dosage in the range of about 2 to 6U and to at least one muscle of the wrist/forearm selected from the group of Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and Pronator Teres (PT) at a dosage in the range of 4 to 16U per muscle, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dosage of about 20U and to at least one muscle of the shoulder at a dosage of about 15U.

In a further embodiment of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus (PQ) and supinator muscles at a dose of about 2.5U and to at least one muscle of the wrist/forearm selected from the group of Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and Pronator Teres (PT) at a dose of about 10U per muscle, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In one aspect of the invention, a botulinum neurotoxin for use in treating upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 4, 5, 6 or 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), Flexor Carpi Ulnaris (FCU) and tergum circus immutatus (PT), and wherein the dose range administered to the Extensor Carpi Ulnaris (ECU), the Extensor Carpi Radialis (ECR), the tergum quadratus (PQ) and the tergum is from about 2 to 6U, the dose range administered to the Flexor Carpi Radialis (FCR), the Flexor Carpi Ulnaris (FCU) and the tergum circus immutatus (PT) is from about 4 to 16U per muscle, and wherein the botulinum neurotoxin is administered at a dose of at least one muscle of about 20U, and applied to at least one muscle of the shoulder in a dose of about 15U.

In a preferred embodiment of the present invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 5 muscles of the forearm/wrist selected from the group of Extensor Carpi Radialis (ECR), flexor supinator, Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and flexor circumflex (PT), and wherein the dose range administered to the Extensor Carpi Radialis (ECR) and supinator is from about 2 to 6U, and the dose range administered to the Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and the circumflex (PT) is from about 4 to 16U per muscle, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In a further aspect of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 4, 5, 6 or 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), Flexor Carpi Ulnaris (FCU) and teres Pronator (PT), and wherein the dose range administered to the Extensor Carpi Ulnaris (ECU), the Extensor Carpi Radialis (ECR), the teres pronator cruris (PQ) and the tergitus is about 2 to 6U, the dose range administered to the Flexor Carpi Radialis (FCR), the Flexor Carpi Ulnaris (FCU) and the tergitus Pronator (PT) is about 4 to 16U per muscle, and wherein the botulinum neurotoxin is administered at a dose of at least one muscle of about 20U, and is administered to at least one muscle of the shoulder in a dose of about 15U, and wherein the botulinum neurotoxin is not administered to Extensor Digitorum Communis (EDC), biceps brachii, deltoid, and teres major.

In a further preferred embodiment of the invention, the botulinum neurotoxin for use in the treatment of upper limb tremors is applied to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is applied to 5 muscles of the forearm/wrist selected from the group of Extensor Carpi Radialis (ECR), Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and teres Pronator (PT), and wherein the dose administered to Extensor Carpi Radialis (ECR) and supinator muscles ranges from about 2 to 6U, the dosage administered to the Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU), and the teres Pronator (PT) ranges from about 4 to 16U per muscle, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow in a dose of about 20U and to at least one muscle of the shoulder in a dose of about 15U; and wherein the botulinum neurotoxin is not administered to Extensor Digitorum Communis (EDC), biceps brachii, deltoid and great muscle.

In a preferred embodiment of the present invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 4, 5, 6 or 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), extensor carpi radialis, flexor carpi ulnaris (FCR), Flexor Carpi Ulnaris (FCU) and round Pronator (PT), and wherein the dose range for the Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator is about 2.5 to 5U, the dose range for the flexor carpi radialis, Flexor Carpi Ulnaris (FCU) and round Pronator (PT) is about 5 to 15U per muscle, and wherein the botulinum neurotoxin is administered in a dose of at least one muscle of about 20U, and applied to at least one muscle of the shoulder in a dose of about 15U.

In a preferred embodiment of the present invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 5 muscles of the forearm/wrist selected from the group of Extensor Carpi Radialis (ECR), flexor supinator, Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and flexor circumflex (PT), and wherein the dose range administered to the Extensor Carpi Radialis (ECR) and supinator is from about 2 to 5U, and the dose range administered to the Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and the circumflex (PT) is from about 4 to 16U per muscle, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In a preferred embodiment of the present invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 4, 5, 6 or 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), extensor carpi radialis, flexor carpi ulnaris (FCR), Flexor Carpi Ulnaris (FCU) and round Pronator (PT), and wherein the dose range for the Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator is about 2.5 to 5U, the dose range for the flexor carpi radialis, Flexor Carpi Ulnaris (FCU) and round Pronator (PT) is about 5 to 15U per muscle, and wherein the botulinum neurotoxin is administered in a dose of at least one muscle of about 20U, and is administered to at least one muscle of the shoulder in a dose of about 15U, and wherein the botulinum neurotoxin is not administered to Extensor Digitorum Communis (EDC), biceps brachii, deltoid, and teres major.

In a further preferred embodiment of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 5 muscles of the forearm/wrist selected from the group of extensor carpi radialis, flexor volvulus, flexor radiocarpus radialis (FCR), Flexor Carpus Ulnaris (FCU) and detrusor (PT), and wherein the dose range administered to the Extensor Carpus Radialis (ECR) and the flexor volvulus is from about 2 to 5U, the dose range administered to the Flexor Carpus Radialis (FCR), the Flexor Carpus Ulnaris (FCU) and the detrusor (PT) is from about 4 to 16U per muscle, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U, and wherein the botulinum neurotoxin is not administered to Extensor Digitorum Communis (EDC), biceps brachii, deltoid and great muscle.

In a particularly preferred embodiment of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 4, 5, 6 or 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), extensor carpi radialis (FCR), Flexor Carpi Ulnaris (FCU) and tergum (PT), and wherein the dose range for the Extensor Carpi Ulnaris (ECU), flexor carpi radialis (ECR), flexor carpi Pronatus (PQ) and tergum is from about 2.5 to 5U, the dose range for the Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and tergum (PT) is from about 5 to 15U per muscle, and wherein the botulinum neurotoxin is administered at a dose of at least one muscle of about 20U, and is administered to at least one muscle of the shoulder at a dose of about 15U, and wherein the total dose of botulinum neurotoxin administered to a muscle of the forearm/carpus does not exceed 65U.

In a further preferred embodiment of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 5 muscles of the forearm/wrist selected from the group of extensor carpi radialis, flexor volvulus, Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and teres Pronator (PT), and wherein the dose range administered to the Extensor Carpi Radialis (ECR) and the tergitalis is from about 2 to 5U, the dose range administered to the Flexor Carpi Radialis (FCR), the Flexor Carpi Ulnaris (FCU) and the tergitalis (PT) is from about 4 to 16U per muscle, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U, and wherein the total dose of botulinum neurotoxin administered to a muscle of the forearm/carpus does not exceed 65U.

In another aspect of the present invention, a botulinum neurotoxin for use in treating upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ), supinator, flexor carpi radialis Flexor (FCR), Flexor Carpi Ulnaris (FCU) and round Pronator (PT), wherein the dose administered to the Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator is about 2.5U and the dose administered to the Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and round Pronator (PT) is about 10U per muscle; and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

In a further aspect of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), extensor carpi radialis, quadratus Pronator (PQ), supinator, flexor carpi radialis Flexor (FCR), Flexor Carpi Ulnaris (FCU) and round Pronator (PT), and wherein the dose administered to the Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ) and supinator is about 2.5U, the dose administered to the flexor carpi radialis, Flexor Carpi Ulnaris (FCU) and round Pronator (PT) is about 10U per muscle, and wherein the botulinum neurotoxin is administered to at least one muscle of the elbow at a dose of about 20U, and is administered to at least one muscle of the shoulder in a dose of about 15U, and wherein the botulinum neurotoxin is not administered to Extensor Digitorum Communis (EDC), biceps brachii, deltoid, and teres major.

In a particularly preferred embodiment of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to 7 muscles of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), quadratus Pronator (PQ), supinator, Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU) and round Pronator (PT), and wherein the dose administered to the Extensor Carpi Ulnaris (ECU), the Extensor Carpi Radialis (ECR), the quadratus Pronator (PQ) and the supinator is about 2.5U, the dose administered to the Flexor Carpi Radialis (FCR), the Flexor Carpi Ulnaris (FCU) and the round Pronator (PT) is about 10U per muscle, and wherein the botulinum neurotoxin is administered in a dose of about 20U to at least one muscle of the arm, and is administered to at least one muscle of the shoulder at a dose of about 15U, and wherein the total dose of botulinum neurotoxin administered to a muscle of the forearm/carpus does not exceed 40U.

In the context of the present invention, upper limb tremor may be tremor of a patient suffering from essential tremor, tremor due to a neurodegenerative disease such as parkinson's disease, dystonic tremor, cerebellar tremor and any other tremor of the upper limb (e.g. musicial tremor, houms tremor, neuropathic tremor, task or location specific tremor, resting tremor, action tremor, etc.).

Further, tremor symptoms and syndromes of childhood and adolescence are also within the context of the present invention. In these patients, the administration of the therapeutic agent should be adjusted according to the body weight of the child receiving the treatment. The adapted dose is in the range of 1-8U/kg BW per upper limb (140U maximum).

In general, it is contemplated according to the present invention that the botulinum neurotoxin for use is administered to the forearm/carpus muscle in a total dose ranging between 30U and 65U. In a more specific embodiment, it is contemplated that the botulinum neurotoxin for use is administered to the forearm/carpus muscle at a total dose ranging between 40U to 65U. In a particular embodiment of the invention, the botulinum neurotoxin for use is administered to the forearm/carpus muscle in a total dose not exceeding 65U. In a further embodiment of the invention, the botulinum neurotoxin for use is administered to the forearm/carpus muscle in a total dose of not more than 40U. In a preferred embodiment of the present invention, the botulinum neurotoxin for use is administered to the extensor and flexor muscles of the carpus/forearm at a dosage ratio in the range of 1:2 to 1: 6.

In general, it is contemplated in accordance with the present invention that the botulinum neurotoxin for use is applied to at least one muscle of each of the forearm/wrist, elbow and shoulder.

In a further aspect of the invention, Extensor Carpi Radialis (ECR) may be divided into extensor carpi radialis longus and extensor carpi radialis brevis.

In a further embodiment of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), extensor pollicis Profundalis (PQ), extensor radialis longus, extensor radialis brevis and supinator, and wherein the botulinum neurotoxin is administered to the Extensor Carpi Ulnaris (ECU), the extensor pollicis radialis (PQ) and the supinator radialis at a dosage in the range of about 2.5 to 6U and is administered to the extensor carpi radialis longus at a dosage in the range of between 1.25U and 3U and to the extensor carpi radialis brevis at a dosage in the range of between 1.25U and 3U and is administered to the extensor carpi brevis/forearm at a dosage in the range of 4 to 16U per muscle, At least one muscle of the group of ulnar carpal flexor muscle (FCU) and Pronator Teres (PT), and wherein the botulinum neurotoxin is applied to the at least one muscle of the elbow at a dose of about 20U and to the at least one muscle of the shoulder at a dose of about 15U.

In a further embodiment of the invention, a botulinum neurotoxin for use in the treatment of upper extremity tremor is administered to at least one muscle of the forearm/wrist, elbow and shoulder, wherein the botulinum neurotoxin is administered to at least one muscle of the forearm/wrist selected from the group of Extensor Carpi Ulnaris (ECU), extensor quadratus (PQ), extensor radialis longus, extensor radialis brevis and supinator muscles, wherein the botulinum neurotoxin is administered to the Extensor Carpi Ulnaris (ECU), the extensor quadratus (PQ) and the supinator radialis at a dosage in the range of about 2.5 to 5U, to the extensor carpi radialis longus at a dosage of 1.25U and to the extensor radialis brevis at a dosage of 1.25U, and to at least one muscle of the group of flexor radialis, flexor ulnar (FCU) and supinator teres (PT) at the wrist/forearm at a dosage in the range of 4 to 16U per muscle, and wherein the botulinum neurotoxin is applied to at least one muscle of the elbow at a dose of about 20U and to at least one muscle of the shoulder at a dose of about 15U.

According to the invention, the botulinum neurotoxin for use is not administered to a muscle of the forearm/carpus selected from the group consisting of: superficial and deep flexors of the fingers, long palmaris, long flexors of the thumb, short and extensors of the thumb, abductor of the thumb, palmaris of the thumb, short abductor of the thumb, short flexors of the thumb, abductor of the thumb, dorsalis interosseus, lumbricalis, palmaris of the little finger, abductor of the little finger and flexors of the little finger. In a preferred embodiment of the present invention, the botulinum neurotoxin for use is not applied to the extensor digitorum communis.

In a further embodiment of the invention, the botulinum neurotoxin for use is administered to at least one muscle of the elbow selected from the group of the brachial and triceps brachii muscles. In a preferred embodiment, the botulinum neurotoxin for use is administered to at least one muscle selected from the group of the brachial and triceps muscles of the elbow at a dose of about 20U per muscle. In a particularly preferred embodiment of the present invention, the botulinum neurotoxin for use is applied to the elbow muscle in a total dose of not more than 40U.

In the context of the present invention, the botulinum neurotoxin for use is not applied to a muscle selected from the group consisting of the brachioradial muscle and the ancuous muscle of the elbow. In a further embodiment of the present invention, the botulinum neurotoxin for use is not applied to the biceps brachii muscle.

In a further embodiment of the present invention, the botulinum toxin for use is applied to at least one muscle selected from the group of latissimus dorsi, pectoralis major, supraspinatus and infraspinatus of the shoulder. In a specific embodiment of the present invention, the botulinum toxin for use is administered to at least one muscle selected from the group of latissimus dorsi, pectoralis major, supraspinatus and infraspinatus in the shoulder at a dose of about 15U per muscle. In a further embodiment of the present invention, the botulinum neurotoxin for use is administered to the shoulder muscle, wherein the total dose does not exceed 60U.

In the context of the present invention, the botulinum neurotoxin for use is not administered to a muscle of the shoulder selected from the group consisting of: brachiocephalic, pectoralis minor, subclavian, subscapularis, serratus anterius, levator scapulae, rhomboid and rhomboid, and trapezius. In a further preferred embodiment of the present invention, the botulinum neurotoxin for use is not applied to the deltoid and the great muscle.

In general, it is contemplated in accordance with the present invention that the botulinum neurotoxin for use is applied to muscles of the forearm/wrist, elbow and shoulder at a total dose in the range between 130U and 165U. In a more specific embodiment, it is contemplated that the botulinum neurotoxin for use is administered to muscles of the forearm/wrist, elbow and shoulder at a total dose ranging between 140U and 165U. In a preferred embodiment of the invention, the botulinum neurotoxin for use is applied to muscles of the forearm/wrist, elbow and shoulder in a total dose not exceeding 165U. In a further preferred embodiment of the invention, the botulinum neurotoxin for use is applied to the muscles of the forearm/wrist, elbow and shoulder in a total dose of not more than 140U. If the botulinum neurotoxin is to be used bilaterally, the corresponding dose is to be adjusted accordingly and doubled compared to a unilateral treatment. Generally, it is contemplated according to the present invention that the botulinum neurotoxin for use is bilaterally applied to the muscles of the forearm/wrist, elbow and shoulder at a total dose ranging between 260U and 330U. In a more specific embodiment, it is contemplated that the botulinum neurotoxin for use is bilaterally applied to muscles of the forearm/wrist, elbow and shoulder at a total dose ranging between 280U and 330U. In a preferred embodiment of the invention, the botulinum neurotoxin for use is applied bilaterally to muscles of the forearm/wrist, elbow and shoulder at a total dose of not more than 330U. In a further preferred embodiment of the invention, the botulinum neurotoxin for use is applied bilaterally to the muscles of the forearm/wrist, elbow and shoulder in a total dose of not more than 280U.

In a preferred embodiment, the botulinum neurotoxin for use is applied to at least one muscle of the forearm/wrist, at least one muscle of the elbow and at least one muscle of the shoulder according to the following dosing regimens:

about 2.5-5.0U of Extensor Carpi Ulnaris (ECU)

Extensor Carpi Radialis (ECR) of about 2.5-5.0U

About 5-15U of Flexor Carpi Radialis (FCR)

Flexor Carpi Ulnaris (FCU) about 5-15U

Round muscle of Pronation (PT) about 5-15U

The amount of the premolar muscle (PQ) is about 2.5-5.0U

Supinator muscle about 2.5-5.0U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

In a particularly preferred embodiment, the botulinum neurotoxin for use is applied to 4, 5, 6 or 7 muscles of the forearm/wrist, 2 muscles of the elbow and 4 muscles of the shoulder according to a dosing regimen:

about 2.5-5.0U of Extensor Carpi Ulnaris (ECU)

Extensor Carpi Radialis (ECR) of about 2.5-5.0U

About 5-15U of Flexor Carpi Radialis (FCR)

Flexor Carpi Ulnaris (FCU) about 5-15U

Round muscle of Pronation (PT) about 5-15U

The amount of the premolar muscle (PQ) is about 2.5-5.0U

Supinator muscle about 2.5-5.0U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

In a particularly preferred embodiment, the botulinum neurotoxin for use is applied to 5 muscles of the forearm/wrist, 2 muscles of the elbow and 4 muscles of the shoulder according to a dosing regimen:

extensor Carpi Radialis (ECR) of about 2.5-5.0U

About 5-15U of Flexor Carpi Radialis (FCR)

Flexor Carpi Ulnaris (FCU) about 5-15U

Round muscle of Pronation (PT) about 5-15U

Supinator muscle about 2.5-5.0U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

In a further preferred embodiment, the botulinum neurotoxin for use is applied to at least one muscle of the forearm/wrist, at least one muscle of the elbow and at least one muscle of the shoulder according to a dosing regimen:

extensor Carpi Ulnaris (ECU) of about 2.5U

Extensor Carpi Radialis (ECR) of about 2.5U

Flexor Carpi Radialis (FCR) about 10U

Flexor Carpi Ulnaris (FCU) of about 10U

Round muscle (PT) of about 10U

The anterior quadratus (PQ) is about 2.5U

Supinator muscle about 2.5U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

In a particularly preferred embodiment, the botulinum neurotoxin for use is applied to 7 muscles of the forearm/wrist, 2 muscles of the elbow and 4 muscles of the shoulder according to a dosing regimen:

extensor Carpi Ulnaris (ECU) of about 2.5U

Extensor Carpi Radialis (ECR) of about 2.5U

Flexor Carpi Radialis (FCR) about 10U

Flexor Carpi Ulnaris (FCU) of about 10U

Round muscle (PT) of about 10U

The anterior quadratus (PQ) is about 2.5U

Supinator muscle about 2.5U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

In an alternative embodiment of the present invention, the botulinum neurotoxin for use is applied to at least one muscle of the forearm/wrist, at least one muscle of the elbow and at least one muscle of the shoulder according to a dosing regimen:

about 2.5-5.0U of Extensor Carpi Ulnaris (ECU)

Extensor Carpi Radialis (ECR) of about 2.5-5.0U

About 5-15U of Flexor Carpi Radialis (FCR)

Flexor Carpi Ulnaris (FCU) about 5-15U

Round muscle of Pronation (PT) about 5-15U

The amount of the premolar muscle (PQ) is about 2.5-5.0U

Supinator muscle about 2.5-5.0U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

Deltoid muscle about 10-15U

In a further alternative embodiment of the invention, the botulinum neurotoxin for use is applied to 4, 5, 6 or 7 muscles of the forearm/wrist, 2 muscles of the elbow and 4 muscles of the shoulder according to a dosing schedule:

about 2.5-5.0U of Extensor Carpi Ulnaris (ECU)

Extensor Carpi Radialis (ECR) of about 2.5-5.0U

About 5-15U of Flexor Carpi Radialis (FCR)

Flexor Carpi Ulnaris (FCU) about 5-15U

Round muscle of Pronation (PT) about 5-15U

The amount of the premolar muscle (PQ) is about 2.5-5.0U

Supinator muscle about 2.5-5.0U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

Deltoid muscle about 10-15U

In a further alternative embodiment of the invention, the botulinum neurotoxin for use is applied to 5 muscles of the forearm/wrist, 2 muscles of the elbow and 4 muscles of the shoulder according to a dosing schedule:

extensor Carpi Radialis (ECR) of about 2.5-5.0U

About 5-15U of Flexor Carpi Radialis (FCR)

Flexor Carpi Ulnaris (FCU) about 5-15U

Round muscle of Pronation (PT) about 5-15U

Supinator muscle about 2.5-5.0U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

Deltoid muscle about 10-15U

In a further alternative embodiment of the present invention, the botulinum neurotoxin for use is applied to at least one muscle of the forearm/wrist, at least one muscle of the elbow and at least one muscle of the shoulder according to a dosing regimen:

extensor Carpi Ulnaris (ECU) of about 2.5U

Extensor Carpi Radialis (ECR) of about 2.5U

Flexor Carpi Radialis (FCR) about 10U

Flexor Carpi Ulnaris (FCU) of about 10U

Round muscle (PT) of about 10U

The anterior quadratus (PQ) is about 2.5U

Supinator muscle about 2.5U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

Deltoid muscle about 10-15U

In a further alternative embodiment of the invention, the botulinum neurotoxin for use is applied to 7 muscles of the forearm/wrist, 2 muscles of the elbow and 5 muscles of the shoulder according to a dosing schedule:

extensor Carpi Ulnaris (ECU) of about 2.5U

Extensor Carpi Radialis (ECR) of about 2.5U

Flexor Carpi Radialis (FCR) about 10U

Flexor Carpi Ulnaris (FCU) of about 10U

Round muscle (PT) of about 10U

The anterior quadratus (PQ) is about 2.5U

Supinator muscle about 2.5U

Humerus muscle of about 20U

Triceps brachii muscle about 20U

About 15U of latissimus dorsi

Pectoralis major muscle about 15U

Supraspinatus approximately 15U

Infraspinatus muscle about 15U

Deltoid muscle about 10-15U

In the context of the present invention, the botulinum neurotoxin for use may be administered either unilaterally or bilaterally. In a preferred embodiment, the botulinum neurotoxin for use is administered bilaterally.

In a particular embodiment of the invention, the botulinum neurotoxin for use is applied as an aqueous reconstitution solution to the muscles of the wrist/forearm, elbow and shoulder at a concentration in the range of 20 to 80U/mL. In a preferred embodiment of the present invention, the botulinum neurotoxin for use is applied as an aqueous solution to the muscles of the wrist/forearm, elbow and shoulder at a concentration in the range of 25 to 60U/mL. In a particularly preferred embodiment, the botulinum neurotoxin for use is applied as an aqueous solution to the muscles of the wrist/forearm, elbow and shoulder at a concentration of 25U/mL. It is generally understood that botulinum neurotoxin can be prepared as a lyophilizate (lyophilizate) comprising the active agent and a pharmaceutically acceptable excipient. Prior to use, the lyophilizate is reconstituted by the addition of an appropriate amount of physiological saline or other suitable reconstitution medium to provide a reconstituted botulinum neurotoxin solution of the desired concentration. Alternatively, the botulinum neurotoxin for use may be provided as a pre-filled syringe as disclosed in, for example, WO2016/102068, WO 2016/124213, or WO 2017/220553. The latter application form avoids any reconstitution step and allows the manufacture and provision of botulinum neurotoxin which has been suitably concentrated.

In the context of the present invention, the botulinum neurotoxin for use is applied to the wrist/forearm, elbow and shoulder muscles of the arm in a total volume of no more than 6.6mL per arm. In a further embodiment of the present invention, the botulinum neurotoxin for use is administered to each muscle at 0.1-0.8mL per muscle.

Generally, the botulinum neurotoxin for use is applied to each muscle in connection with muscle dissection. The person skilled in the art knows the method for applying the botulinum neurotoxin for use in an anatomical position of a motor unit. The number of injection points per muscle will vary depending on the anatomical size, shape, physiological strength and function of the muscle, and the location and extent of the motor endplates of the nerves that will be affected by the botulinum neurotoxin. Anatomical atlases are used to define the location and extent of injections given to each muscle. The injection of botulinum neurotoxin for use can also be guided by using EMG, ultrasound or electrical stimulation means of the corresponding muscle.

The number of injection sites is generally determined by the corresponding muscle size, and one skilled in the art knows how to administer botulinum neurotoxin to a particular muscle. In the context of the present invention, botulinum neurotoxin is injected into a muscle of the wrist/forearm selected from the group of Extensor Carpi Ulnaris (ECU), Extensor Carpi Radialis (ECR), Flexor Carpi Radialis (FCR), Flexor Carpi Ulnaris (FCU), teres circumflex (PT), quadratus Pronator (PQ) and supinator by using one injection site per muscle. Botulinum neurotoxin is injected into a muscle selected from the group consisting of the brachial muscle and the triceps brachii muscle at the elbow using two sites per muscle. Botulinum neurotoxin is injected into a muscle selected from latissimus dorsi and pectoralis major in the shoulder using two sites per muscle. Botulinum neurotoxin is injected into a muscle selected from supraspinatus and infraspinatus of the shoulder using three sites per muscle.

Severity of tremor was assessed according to the present invention by the essential tremor assessment scale (TETRAS). This assessment uses validated clinical scales specifically designed to assess the severity of ET. Ratings of 0-4 are made at half-point intervals for the head when standing, face including jaw, vocal cords, upper limb, and lower limb on the worse side. The scale focuses on the assessment of upper limb action tremor using the following subcategories: writing on the dominant side only; separate evaluations of both sides were performed for the following cases: using the posture of the arm extended forward and the position of the flapping flash, the dynamics of the experiment performed with the fingers against the nose, drawing an archimedean spiral, and point approximation (where the pen is brought as close as possible to a point on a piece of paper without touching it). The TETRAS system has anchor points that span a greater range of tremor amplitudes (>20 cm-4 scale), making it suitable for evaluating severe ET patients. The total score of all the items of tetrars was evaluated to characterize the severity of tremors. The upper limb athletic performance score is rated by an experienced physician according to the task performed by the subject (as indicated by the investigator). The phenotype scale allows for 0.5 point increments in the score. The 0.5 point increments of upper limb tremor rating are defined by a specific range of tremor amplitudes. The minimum detectable change in the TETRAS performance scale is 8.9% of the baseline measurement [ Voller et al, Mov dis.2015 ].

In the context of the present invention, botulinum neurotoxin for use improves the severity of tremors by at least 9.0% according to the TETRAS upper limb motor performance score scale rated by the investigator. In a preferred embodiment, the botulinum neurotoxin for use improves the severity of tremors by at least 15% according to the tetra s upper limb motor performance score scale rated by the investigator. In a more preferred embodiment, the botulinum neurotoxin for use improves the severity of tremors by at least 25% according to the tetra s upper limb motor performance score scale rated by the investigator. In a most preferred embodiment, the botulinum neurotoxin for use improves the severity of tremors by at least 40% according to the tetra s upper limb motor performance score scale rated by the investigator.

Alternatively, the severity of tremor may be assessed according to the present invention by using a standardized computerized kinematic tremor analysis. This analysis assesses tremor intensity by measuring the amplitude of angular tremor associated with functional muscle groups at shoulder (flexion/extension, adduction/abduction), elbow (flexion/extension) and wrist/forearm level (flexion/extension, radial/ulnar deviation, pronation/supination) for a series of 3 analytical trials for each of the following tasks: I. posture 1: the shoulder is flexed by 90 degrees, and the forearm is stretched forwards and revolved forwards; posture 2: shoulder flexion 90 °, forearm extension forward in neutral position; posture 3: the shoulder is flexed 90 °, the elbow is also flexed, both hands are positioned close to the mouth; IV, load 1: holding an empty plastic cup (33 g); v, load 2: hold the same plastic cup (355ml soda can) with a 1 pound weight; VI, load 3: hold the phone (this will mimic holding a different object). All three load evaluations were performed in a posture with the shoulder flexed 90 ° and the forearm extended forward.

In the context of the present invention, the botulinum neurotoxin for use reduces the maximum logarithmically transformed acceleration hand tremor amplitude at week 4 by at least-0.10 m/s from baseline as compared to placebo²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]). In a preferred embodiment of the invention, the botulinum neurotoxin for use reduces the maximum logarithmically transformed acceleration hand tremor amplitude at week 4 by at least-0.10 m/s from baseline as compared to placebo²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]) And decreased by at least-0.10 m/s at week 6²(least squares [ LS)]Mean difference of botulinum toxinNeurotoxin A and placebo]). In a more preferred embodiment of the invention, the botulinum neurotoxin for use reduces the maximum logarithmically transformed acceleration hand tremor amplitude at week 4 by at least-0.10 m/s from baseline as compared to placebo²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]) At week 6, a reduction of at least-0.10 m/s²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]) And decreased by at least-0.10 m/s at week 8²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]). In a preferred aspect of the invention, the botulinum neurotoxin for use reduces the maximum logarithmically transformed acceleration hand tremor amplitude at week 4 by at least-0.20 m/s from baseline as compared to placebo²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]). In a preferred embodiment of the invention, the botulinum neurotoxin for use reduces the maximum logarithmically transformed acceleration hand tremor amplitude at week 4 by at least-0.20 m/s from baseline as compared to placebo²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]) And decreased by at least-0.20 m/s at week 6²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]). In a more preferred embodiment of the invention, the botulinum neurotoxin for use reduces the maximum log-transformed acceleration hand tremor amplitude at week 4 by at least-0.20 m/s from baseline as compared to placebo²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]) And decreased by at least-0.20 m/s at week 6²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]) And decreased by at least-0.20 m/s at week 8²(least squares [ LS)]Mean difference botulinum neurotoxin A and placebo]). In a further aspect of the invention, the botulinum neurotoxin for use reduces the maximum carpal angle tremor amplitude at week 4 by-0.7 degrees from baseline (LS mean difference botulinum neurotoxin A vs placebo) compared to placebo]). In a preferred embodiment of the invention, the botulinum neurotoxin used reduces the maximum carpal angle tremor amplitude at week 4 by-0.7 degrees from baseline (LS mean difference botulinum neurotoxin A vs placebo) compared to placebo]) And decreases at week 6Lower by-0.7 degree (LS mean difference botulinum neurotoxin A vs placebo)]). In a more preferred embodiment of the invention, the botulinum neurotoxin for use reduces the maximum carpal angle tremor amplitude at week 4 by-0.7 degrees from baseline (LS mean difference botulinum neurotoxin A vs placebo)]) Decrease by-0.7 degree at week 6 (LS mean Difference botulinum neurotoxin A vs placebo)]) And decreased by-0.7 degrees at week 8 (LS mean Difference [ botulinum neurotoxin A vs placebo)]). In a further preferred aspect of the invention, the botulinum neurotoxin for use reduces the maximum carpal tremor amplitude at week 4 by-0.14 degrees from baseline (LS mean difference botulinum neurotoxin A vs placebo) compared to placebo]). In a preferred embodiment of the invention, the botulinum neurotoxin used reduces the maximum carpal angle tremor amplitude at week 4 by-0.14 degrees from baseline (LS mean difference botulinum neurotoxin A vs placebo) compared to placebo]) And decreased by-0.14 degrees at week 6 (LS mean Difference [ botulinum neurotoxin A vs placebo)]). In a more preferred embodiment of the invention, the botulinum neurotoxin for use reduces the maximum carpal angle tremor amplitude at week 4 by-0.14 degrees from baseline (LS mean difference botulinum neurotoxin A vs placebo)]) Decrease by-0.14 degrees at week 6 (LS mean Difference botulinum neurotoxin A vs placebo)]) And decreased by-0.14 degrees at week 8 (LS mean Difference [ botulinum neurotoxin A vs placebo)])。

In vivo assays for assessing botulinum neurotoxin biological activity include mouse LD50 assays and ex vivo mouse diaphragm assays as described by Pearce et al (Pearce 1994, Toxicol. appl. Pharmacol.128:69-77) and Dressler et al (Dressier 2005, Mov. Disord.20:1617-1619, Keller 2006, Neuroscience 139:629-637), or cell-based assays as described in WO2009/114748, WO2014/207109 or WO 2013/049508. Biological activity is usually expressed in mouse units (U). As used herein, 1U is the amount of the neurotoxic component of botulinum neurotoxin that kills 50% of a given population of mice, i.e., mouse i.p. ld50, after intraperitoneal injection. Particularly useful methods for determining the biological activity of a botulinum neurotoxin are as disclosed, for example, in WO2009/114748, WO 2013/049508 or WO2014/207109The cell-based assay of (1). The activity results obtained with such cell-based assays correspond to the activity values obtained in the mouse i.p. ld50 assay. Using conversion rates known to those skilled in the art, a protein directed against a botulinum serotype a formulation such as the commercially available Incobotulinumtoxin a (botulinum toxin serotype a without complexing proteins,merz Pharmaceuticals GmbH)) or Onabotulinumtoxin a (botulinum toxin serotype a containing complexing proteins,aller gan company) to values for other toxins. For example, by multiplying the dose of Incobotulinumtoxin A or Onobotulinumtoxin A by a factor of 2.5 to 5, Abobotulinumtoxin A (botulinum toxin serotype A containing complexing proteins,ipsen Biopharm Limited). Rimabotulinumtoxin B (botulinum toxin serotype B, solstices neurosciens/US worldmed LLC).

In the context of the present invention, the term "botulinum neurotoxin" refers to a native neurotoxin obtainable from the bacterium clostridium botulinum or a neurotoxin obtainable from an alternative source, including from recombinant techniques or from genetic or chemical modifications. In particular, botulinum neurotoxin has endopeptidase activity. In the context of the present invention, the terms "botulinum toxin" and "botulinum neurotoxin" are used synonymously and interchangeably.

In a particular embodiment, the botulinum neurotoxin according to the invention is a botulinum neurotoxin complex.

In the context of the present invention, the terms "toxin complex" or "botulinum neurotoxin complex" are interchangeable and refer to a high molecular weight complex comprising a neurotoxic component of approximately 150kDa and additionally comprising non-toxic proteins of Clostridium botulinum including hemagglutinin and non-hemagglutinin proteins (Sakaguchi 1983; Sugiyama 1980). When a botulinum toxin is released from a lysed clostridial culture, it typically associates with other bacterial proteins, which together form a toxin complex. Such complexes typically contain additional so-called "non-toxic" proteins, which are referred to herein as "complex proteins" or "bacterial proteins. The complex of the neurotoxic component and the bacterial protein is called the "clostridium botulinum toxin complex" or "botulinum toxin complex". The molecular weight of such complexes may vary from about 300,000 to about 900,000 Da. It is commercially available as botulinum toxin a protein complex, for example under the trade name botox (allergan inc) or DYSPORT (ipsen ltd).

In the context of the present invention, the term "neurotoxic component" or "neurotoxin component" as used throughout the specification relates to a subunit of a botulinum toxin complex, which subunit has neurotoxic activity and a molecular weight of approximately 150kDa in serotype a. Unlike toxin complexes, the neurotoxic component in its isolated and pure form (i.e., lacking any complex clostridial proteins) is acid labile and does not resist the aggressive environment in the gastrointestinal tract. A method for purifying and manufacturing the neurotoxic component of a botulinum neurotoxin is shown in US 8,398,998. Highly pure neurotoxic components free of any complexing proteins are available, for example, from Merz Pharmaceuticals GmbH of Frankfurt

The term "neurotoxic component" also includes functional homologues found in other serotypes of clostridium botulinum.

In a particular embodiment, the botulinum neurotoxin according to the present invention is a neurotoxic component of a botulinum neurotoxin complex, wherein the neurotoxic component is devoid of any other protein component of a clostridium botulinum neurotoxin complex.

In the context of the present invention, the term "lacking any other protein component of the clostridium botulinum neurotoxin complex" refers to any non-toxic protein free of clostridium botulinum, such as a hemagglutinin protein.

In a particular embodiment, the botulinum neurotoxin according to the invention is selected from the group of different serotypes comprising: botulinum neurotoxin serotype A (BoNT/A), botulinum neurotoxin serotype B (BoNT/B), botulinum neurotoxin serotype C1(BoNT/C1), botulinum neurotoxin serotype D (BoNT/D), botulinum neurotoxin serotype E (BoNT/E), botulinum neurotoxin serotype F (BoNT/F) or botulinum neurotoxin serotype G (BoNT/G). The botulinum neurotoxin, in particular the light chain and the heavy chain thereof, can be derived from one of the different serotypes on the antigen of the botulinum neurotoxin indicated above. In one aspect, the light chain and heavy chain of the botulinum neurotoxin is a light chain and heavy chain of a botulinum neurotoxin selected from the group consisting of: BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F or BoNT/G. In another aspect, a polynucleotide encoding a botulinum neurotoxin of the present invention comprises a nucleic acid sequence as set forth in SEQ ID NO 1(BoNT/A), SEQ ID NO 3(BoNT/B), SEQ ID NO 5(BoNT/C1), SEQ ID NO 7(BoNT/D), SEQ ID NO 9(BoNT/E), SEQ ID NO 11(BoNT/F), or SEQ ID NO 13 (BoNT/G). Moreover, polynucleotides comprising a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO:2(BoNT/A), SEQ ID NO:4(BoNT/B), SEQ ID NO:6(BoNT/C1), SEQ ID NO:8(BoNT/D), SEQ ID NO:10(BoNT/E), SEQ ID NO:12(BoNT/F) or SEQ ID NO:14(BoNT/G) are encompassed in one aspect. Further encompassed in one aspect are means and methods of the invention for producing a botulinum neurotoxin comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO 2(BoNT/A), SEQ ID NO 4(BoNT/B), SEQ ID NO 6(BoNT/C1), SEQ ID NO 8(BoNT/D), SEQ ID NO 10(BoNT/E), SEQ ID NO 12(BoNT/F) or SEQ ID NO 14 (BoNT/G).

In another aspect, the polynucleotide encoding a botulinum neurotoxin of the present invention is a variant of the aforementioned polynucleotides comprising one or more nucleotide substitutions, deletions and/or additions, which in yet another aspect can produce a polypeptide having one or more amino acid substitutions, deletions and/or additions. Moreover, variant polynucleotides of the invention should in another aspect comprise a variant nucleic acid sequence which is at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a nucleic acid sequence as set forth in any one of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13 or 15, or a variant nucleic acid sequence which encodes an amino acid sequence which is at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence as set forth in any one of SEQ ID NOs 2,4, 6, 8, 10, 12, 14 or 16. The term "identical" as used herein refers to sequence identity, which is characterized by determining the number of identical amino acids between two nucleic acid sequences or two amino acid sequences, wherein the sequences are aligned for the highest order match. It can be calculated using published techniques or methods programmed into computer programs, such as BLASTP, BLASTN or FASTA (Altschul 1990, J Mol Biol 215, 403). Percent identity values are in one aspect calculated over the entire amino acid sequence. There are a number of programs based on various algorithms for technicians to compare different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. For sequence alignment, the programs PileUp (Higgins 1989, CABIOS 5,151) or the programs Gap and BestFit (Needleman 1970, J Mol Biol 48; 443; Smith 1981, Adv Appl Math 2,482), which are part of the GCG software package (Genetics Computer Group 1991,575Science Drive, Madison, Wisconsin, USA 53711), can be used. The sequence identity values listed above in percent (%) in another aspect of the invention will utilize the following settings over the entire sequence area using the program GAP: empty bit weight: 50, length weight: 3, average matching: 10.000 and average mismatches: 0.000, these settings should always be used as standard settings for sequence alignment unless otherwise stated. In one aspect, each of the foregoing variant polynucleotides encodes a polypeptide that retains one or more biological properties of the corresponding botulinum neurotoxin (i.e., BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, or BoNT/G), while in another aspect each of the foregoing variant polynucleotides encodes a polypeptide that retains all biological properties of the corresponding botulinum neurotoxin (i.e., BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, or BoNT/G). It will be appreciated by those skilled in the art that full biological activity is retained only after proteolytic activation, although it is envisaged that the unprocessed precursor may perform some biological function or be partially active. "biological property" as used herein refers to (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of a protein involved in synaptic vesicle membrane fusion. In a further aspect, the variant polynucleotides may encode botulinum neurotoxins with improved or altered biological properties, for example, they may comprise a cleavage site that is improved in enzyme recognition or improved in receptor binding or any other property specified above.

In a particular embodiment of the invention, the botulinum neurotoxin for use in the treatment of upper extremity tremors is administered together with at least one standard therapeutic agent selected from propranolol, paminone, any other antiepileptic drug or calcium channel blocker, or the botulinum neurotoxin is administered in parallel or sequentially with a treatment of deep brain stimulation or magnetic resonance guided high frequency ultrasound, local electrical stimulation, biofeedback, kinematic assessment guided stimulation, anti-tremor devices, anti-tremor smart phone applications, or combinations thereof.

In another aspect, the present invention relates to a pharmaceutical composition comprising a botulinum neurotoxin for use in the treatment of upper limb tremors according to the present invention. To prepare a pharmaceutical preparation comprising a botulinum neurotoxin, the neurotoxin can be formulated by various techniques, depending on the desired application purpose known in the art. For example, the botulinum neurotoxin (biologically active) can be used in a pharmaceutical composition in combination with one or more pharmaceutically acceptable carriers. The one or more pharmaceutically acceptable carriers must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical carrier employed may comprise a solid, gel or liquid. Exemplary solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary liquid carriers are glycerin, phosphate buffered saline solution, water, emulsions, various types of wetting agents, and the like. Suitable carriers include those mentioned above and others well known in the art, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. In one aspect, the pharmaceutical composition may be dissolved in a diluent prior to administration. The diluent is also selected so as not to affect the biological activity of the botulinum neurotoxin product. Examples of such diluents are distilled water or physiological saline. In addition, the pharmaceutical composition or formulation may also include other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. Thus, in one aspect, a formulated botulinum neurotoxin product can be present in a liquid or lyophilized form. In one aspect, it may be present with glycerol, a protein stabilizer (HSA), or a non-protein stabilizer such as polyvinylpyrrolidone (PVP), hyaluronic acid, or a free amino acid such as methionine or histidine. In one aspect, suitable non-proteinaceous stabilizing agents are disclosed in WO 2005/007185, WO 2006/020208, WO2018/135722, WO2006/005910, or WO 2012/134240. For example, suitable formulations comprising an HSA-stabilized formulation of a botulinum neurotoxin according to the present invention are disclosed in US 8,398,998B 2.

In the context of the present invention, the term "comprising" or "includes" means "including but not limited to". The terms are intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but do not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more limiting terms "consisting of … …" and "consisting essentially of … …".

In the context of the present invention, the term "about" refers to the common deviation of the dose of botulinum neurotoxin actually administered to a muscle from the calculated dose. When the botulinum neurotoxin for use is administered in the form of a reconstituted aqueous solution by use of a syringe, it is generally accepted by those skilled in the art that this deviation is +/-10% of the calculated dose.

In a particular embodiment, the pharmaceutical composition comprising a botulinum neurotoxin according to the invention is for use in the treatment of upper limb tremors.

In another aspect, the present invention relates to a method of treating upper limb tremor, wherein the method comprises administering a therapeutically effective amount of a botulinum neurotoxin according to the present invention.

Examples

Example 1: general treatment of upper limb tremor using NT201

NT201 (active ingredient: NT 101, botulinum neurotoxin type A, without complexing proteins, the American adopted name Incobotulinumtoxin A), excipient human serum albumin plus sucrose, provided in vials for reconstitution. For unilateral treatment, two vials containing 100U NT201 were reconstituted with 8.0ml of physiological saline to provide a solution with a concentration of 25U/ml. For bilateral treatment, 3 or 4 vials were reconstituted with 8.0 or 16.0ml of saline to provide the same concentration. No further dilution is required.

For initial treatment, NT201 was injected unilaterally into the muscles of the wrist/forearm and forced into the elbow and shoulder muscles (table below, dosing regimen a). This dosing regimen is applicable to patients with upper limb tremors of any intensity (mild-moderate-significant) and any involvement of the wrist/forearm/elbow/shoulder muscle groups.

Dosing regimen B can be used by an experienced injector, either on one or both sides, initially or after an initial injection is administered to the patient using regimen a. For dosing regimen B, the choice of tremor muscles with a flexible number of muscles in the forearm/wrist was decided by the investigator based on clinical analysis and possibly supported by EMG or any other technical support measures (e.g. kinematic analysis).

The total dose in regimen a was 140U per arm per patient and the maximum allowable dose in regimen B was 165U per arm per patient. In regimen B, there were a minimum of 4 and a maximum of 7 muscles of the forearm tremor muscle that were treated. In dosing regimen a, all 7 forearm/wrist muscles were treated.

In the bilateral treatment, the total dose in regimen A was 280.0U per patient and the total dose in regimen B was 330.0U per patient. For bilateral treatment, the same dosing rules as for unilateral treatment were applied, except that these rules were applied to both treated arms.

TABLE 1

The injection may be guided by EMG, ultrasound or electrical stimulation of the muscle, depending on the decision of the injector. Combinations of these guidance techniques may also be used for one patient, and different techniques may also be used for different muscles.

EXAMPLES treatment of Individual patients

One male patient, bifacial essential tremor, upper limb action tremor with a tetra s score of 3 (amplitude from 5 to less than 10cm), received treatment with NT201 using the following fixed dosing regimen:

after 4 weeks, the tetra rs score for right arm action tremor decreased by 1 point (-33%). After 8 weeks, the tetra s score decreased by 0.5 points (-17%) compared to before injection.

Patients with semi-flexible dosing:

one male patient, mainly with flexion/extension dominated wrist essential tremor, scored 2.5 points, depending on the posture of the arm (tremor amplitude between 3 and less than 5 cm), tetra s, received treatment with NT201 using the following semi-flexible dosing regimen:

after 4 weeks, the teras score decreased by 1 point (-40%). After 8 weeks, the tetra s score was reduced by 0.5 points (-25%) compared to the score immediately prior to injection.

Patients with semi-flexible dosing:

a female patient with flexion/extension dominated essential tremor of the wrist with a rotating component, scored 3 points according to arm posture (tremor amplitude between 5 and less than 10cm), receiving treatment with NT201 using the following semi-flexible dosing regimen:

after 4 weeks, the TERTAS score decreased by 1 point (-33%). After 8 weeks, the tetra s score was still reduced by 1 point (-33%) compared to the score immediately prior to injection.

Sequence listing

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cgtgtttata catttttttc ttcagactat gtaaagaaag ttaataaagc tacggaggca 1800

gctatgtttt taggctgggt agaacaatta gtatatgatt ttaccgatga aactagcgaa 1860

gtaagtacta cggataaaat tgcggatata actataatta ttccatatat aggacctgct 1920

ttaaatatag gtaatatgtt atataaagat gattttgtag gtgctttaat attttcagga 1980

gctgttattc tgttagaatt tataccagag attgcaatac ctgtattagg tacttttgca 2040

cttgtatcat atattgcgaa taaggttcta accgttcaaa caatagataa tgctttaagt 2100

aaaagaaatg aaaaatggga tgaggtctat aaatatatag taacaaattg gttagcaaag 2160

gttaatacac agattgatct aataagaaaa aaaatgaaag aagctttaga aaatcaagca 2220

gaagcaacaa aggctataat aaactatcag tataatcaat atactgagga agagaaaaat 2280

aatattaatt ttaatattga tgatttaagt tcgaaactta atgagtctat aaataaagct 2340

atgattaata taaataaatt tttgaatcaa tgctctgttt catatttaat gaattctatg 2400

atcccttatg gtgttaaacg gttagaagat tttgatgcta gtcttaaaga tgcattatta 2460

aagtatatat atgataatag aggaacttta attggtcaag tagatagatt aaaagataaa 2520

gttaataata cacttagtac agatatacct tttcagcttt ccaaatacgt agataatcaa 2580

agattattat ctacatttac tgaatatatt aagaatatta ttaatacttc tatattgaat 2640

ttaagatatg aaagtaatca tttaatagac ttatctaggt atgcatcaaa aataaatatt 2700

ggtagtaaag taaattttga tccaatagat aaaaatcaaa ttcaattatt taatttagaa 2760

agtagtaaaa ttgaggtaat tttaaaaaat gctattgtat ataatagtat gtatgaaaat 2820

tttagtacta gcttttggat aagaattcct aagtatttta acagtataag tctaaataat 2880

gaatatacaa taataaattg tatggaaaat aattcaggat ggaaagtatc acttaattat 2940

ggtgaaataa tctggacttt acaggatact caggaaataa aacaaagagt agtttttaaa 3000

tacagtcaaa tgattaatat atcagattat ataaacagat ggatttttgt aactatcact 3060

aataatagat taaataactc taaaatttat ataaatggaa gattaataga tcaaaaacca 3120

atttcaaatt taggtaatat tcatgctagt aataatataa tgtttaaatt agatggttgt 3180

agagatacac atagatatat ttggataaaa tattttaatc tttttgataa ggaattaaat 3240

gaaaaagaaa tcaaagattt atatgataat caatcaaatt caggtatttt aaaagacttt 3300

tggggtgatt atttacaata tgataaacca tactatatgt taaatttata tgatccaaat 3360

aaatatgtcg atgtaaataa tgtaggtatt agaggttata tgtatcttaa agggcctaga 3420

ggtagcgtaa tgactacaaa catttattta aattcaagtt tgtatagggg gacaaaattt 3480

attataaaaa aatatgcttc tggaaataaa gataatattg ttagaaataa tgatcgtgta 3540

tatattaatg tagtagttaa aaataaagaa tataggttag ctactaatgc atcacaggca 3600

ggcgtagaaa aaatactaag tgcattagaa atacctgatg taggaaatct aagtcaagta 3660

gtagtaatga agtcaaaaaa tgatcaagga ataacaaata aatgcaaaat gaatttacaa 3720

gataataatg ggaatgatat aggctttata ggatttcatc agtttaataa tatagctaaa 3780

ctagtagcaa gtaattggta taatagacaa atagaaagat ctagtaggac tttgggttgc 3840

tcatgggaat ttattcctgt agatgatgga tggggagaaa ggccactgta a 3891

<210> 2

<211> 1296

<212> PRT

<213> Clostridium botulinum (Clostridium botulinum)

<400> 2

Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly

1 5 10 15

Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro

20 25 30

Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg

35 40 45

Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu

50 55 60

Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr

65 70 75 80

Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu

85 90 95

Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val

100 105 110

Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys

115 120 125

Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr

130 135 140

Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile

145 150 155 160

Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr

165 170 175

Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe

180 185 190

Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu

195 200 205

Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu

210 215 220

Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn

225 230 235 240

Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu

245 250 255

Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys

260 265 270

Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn

275 280 285

Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val

290 295 300

Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys

305 310 315 320

Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu

325 330 335

Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp

340 345 350

Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn

355 360 365

Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr

370 375 380

Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn

385 390 395 400

Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu

405 410 415

Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg

420 425 430

Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys

435 440 445

Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe

450 455 460

Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu

465 470 475 480

Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu

485 490 495

Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro

500 505 510

Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu

515 520 525

Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu

530 535 540

Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu

545 550 555 560

His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu

565 570 575

Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys

580 585 590

Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu

595 600 605

Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr

610 615 620

Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala

625 630 635 640

Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu

645 650 655

Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala

660 665 670

Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys

675 680 685

Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu

690 695 700

Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys

705 710 715 720

Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu

725 730 735

Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn

740 745 750

Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp

755 760 765

Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile

770 775 780

Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met

785 790 795 800

Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys

805 810 815

Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly

820 825 830

Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp

835 840 845

Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser

850 855 860

Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn

865 870 875 880

Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser

885 890 895

Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn

900 905 910

Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu

915 920 925

Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser

930 935 940

Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn

945 950 955 960

Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val

965 970 975

Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu

980 985 990

Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser

995 1000 1005

Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu

1010 1015 1020

Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro

1025 1030 1035 1040

Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys

1045 1050 1055

Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe

1060 1065 1070

Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr

1075 1080 1085

Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr

1090 1095 1100

Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn

1105 1110 1115 1120

Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu

1125 1130 1135

Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser

1140 1145 1150

Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly

1155 1160 1165

Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val

1170 1175 1180

Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala

1185 1190 1195 1200

Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn

1205 1210 1215

Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr

1220 1225 1230

Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly

1235 1240 1245

Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser

1250 1255 1260

Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys

1265 1270 1275 1280

Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu

1285 1290 1295

<210> 3

<211> 3876

<212> DNA

<213> Clostridium botulinum (Clostridium botulinum)

<400> 3

atgccagtta caataaataa ttttaattat aatgatccta ttgataataa taatattatt 60

atgatggagc ctccatttgc gagaggtacg gggagatatt ataaagcttt taaaatcaca 120

gatcgtattt ggataatacc ggaaagatat acttttggat ataaacctga ggattttaat 180

aaaagttccg gtatttttaa tagagatgtt tgtgaatatt atgatccaga ttacttaaat 240

actaatgata aaaagaatat atttttacaa acaatgatca agttatttaa tagaatcaaa 300

tcaaaaccat tgggtgaaaa gttattagag atgattataa atggtatacc ttatcttgga 360

gatagacgtg ttccactcga agagtttaac acaaacattg ctagtgtaac tgttaataaa 420

ttaatcagta atccaggaga agtggagcga aaaaaaggta ttttcgcaaa tttaataata 480

tttggacctg ggccagtttt aaatgaaaat gagactatag atataggtat acaaaatcat 540

tttgcatcaa gggaaggctt cgggggtata atgcaaatga agttttgccc agaatatgta 600

agcgtattta ataatgttca agaaaacaaa ggcgcaagta tatttaatag acgtggatat 660

ttttcagatc cagccttgat attaatgcat gaacttatac atgttttaca tggattatat 720

ggcattaaag tagatgattt accaattgta ccaaatgaaa aaaaattttt tatgcaatct 780

acagatgcta tacaggcaga agaactatat acatttggag gacaagatcc cagcatcata 840

actccttcta cggataaaag tatctatgat aaagttttgc aaaattttag agggatagtt 900

gatagactta acaaggtttt agtttgcata tcagatccta acattaatat taatatatat 960

aaaaataaat ttaaagataa atataaattc gttgaagatt ctgagggaaa atatagtata 1020

gatgtagaaa gttttgataa attatataaa agcttaatgt ttggttttac agaaactaat 1080

atagcagaaa attataaaat aaaaactaga gcttcttatt ttagtgattc cttaccacca 1140

gtaaaaataa aaaatttatt agataatgaa atctatacta tagaggaagg gtttaatata 1200

tctgataaag atatggaaaa agaatataga ggtcagaata aagctataaa taaacaagct 1260

tatgaagaaa ttagcaagga gcatttggct gtatataaga tacaaatgtg taaaagtgtt 1320

aaagctccag gaatatgtat tgatgttgat aatgaagatt tgttctttat agctgataaa 1380

aatagttttt cagatgattt atctaaaaac gaaagaatag aatataatac acagagtaat 1440

tatatagaaa atgacttccc tataaatgaa ttaattttag atactgattt aataagtaaa 1500

atagaattac caagtgaaaa tacagaatca cttactgatt ttaatgtaga tgttccagta 1560

tatgaaaaac aacccgctat aaaaaaaatt tttacagatg aaaataccat ctttcaatat 1620

ttatactctc agacatttcc tctagatata agagatataa gtttaacatc ttcatttgat 1680

gatgcattat tattttctaa caaagtttat tcattttttt ctatggatta tattaaaact 1740

gctaataaag tggtagaagc aggattattt gcaggttggg tgaaacagat agtaaatgat 1800

tttgtaatcg aagctaataa aagcaatact atggataaaa ttgcagatat atctctaatt 1860

gttccttata taggattagc tttaaatgta ggaaatgaaa cagctaaagg aaattttgaa 1920

aatgcttttg agattgcagg agccagtatt ctactagaat ttataccaga acttttaata 1980

cctgtagttg gagccttttt attagaatca tatattgaca ataaaaataa aattattaaa 2040

acaatagata atgctttaac taaaagaaat gaaaaatgga gtgatatgta cggattaata 2100

gtagcgcaat ggctctcaac agttaatact caattttata caataaaaga gggaatgtat 2160

aaggctttaa attatcaagc acaagcattg gaagaaataa taaaatacag atataatata 2220

tattctgaaa aagaaaagtc aaatattaac atcgatttta atgatataaa ttctaaactt 2280

aatgagggta ttaaccaagc tatagataat ataaataatt ttataaatgg atgttctgta 2340

tcatatttaa tgaaaaaaat gattccatta gctgtagaaa aattactaga ctttgataat 2400

actctcaaaa aaaatttgtt aaattatata gatgaaaata aattatattt gattggaagt 2460

gcagaatatg aaaaatcaaa agtaaataaa tacttgaaaa ccattatgcc gtttgatctt 2520

tcaatatata ccaatgatac aatactaata gaaatgttta ataaatataa tagcgaaatt 2580

ttaaataata ttatcttaaa tttaagatat aaggataata atttaataga tttatcagga 2640

tatggggcaa aggtagaggt atatgatgga gtcgagctta atgataaaaa tcaatttaaa 2700

ttaactagtt cagcaaatag taagattaga gtgactcaaa atcagaatat catatttaat 2760

agtgtgttcc ttgattttag cgttagcttt tggataagaa tacctaaata taagaatgat 2820

ggtatacaaa attatattca taatgaatat acaataatta attgtatgaa aaataattcg 2880

ggctggaaaa tatctattag gggtaatagg ataatatgga ctttaattga tataaatgga 2940

aaaaccaaat cggtattttt tgaatataac ataagagaag atatatcaga gtatataaat 3000

agatggtttt ttgtaactat tactaataat ttgaataacg ctaaaattta tattaatggt 3060

aagctagaat caaatacaga tattaaagat ataagagaag ttattgctaa tggtgaaata 3120

atatttaaat tagatggtga tatagataga acacaattta tttggatgaa atatttcagt 3180

atttttaata cggaattaag tcaatcaaat attgaagaaa gatataaaat tcaatcatat 3240

agcgaatatt taaaagattt ttggggaaat cctttaatgt acaataaaga atattatatg 3300

tttaatgcgg ggaataaaaa ttcatatatt aaactaaaga aagattcacc tgtaggtgaa 3360

attttaacac gtagcaaata taatcaaaat tctaaatata taaattatag agatttatat 3420

attggagaaa aatttattat aagaagaaag tcaaattctc aatctataaa tgatgatata 3480

gttagaaaag aagattatat atatctagat ttttttaatt taaatcaaga gtggagagta 3540

tatacctata aatattttaa gaaagaggaa gaaaaattgt ttttagctcc tataagtgat 3600

tctgatgagt tttacaatac tatacaaata aaagaatatg atgaacagcc aacatatagt 3660

tgtcagttgc tttttaaaaa agatgaagaa agtactgatg agataggatt gattggtatt 3720

catcgtttct acgaatctgg aattgtattt gaagagtata aagattattt ttgtataagt 3780

aaatggtact taaaagaggt aaaaaggaaa ccatataatt taaaattggg atgtaattgg 3840

cagtttattc ctaaagatga agggtggact gaataa 3876

<210> 4

<211> 1291

<212> PRT

<213> Clostridium botulinum (Clostridium botulinum)

<400> 4

Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn

1 5 10 15

Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg

20 25 30

Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu

35 40 45

Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly

50 55 60

Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn

65 70 75 80

Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe

85 90 95

Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile

100 105 110

Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu

115 120 125

Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn

130 135 140

Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile

145 150 155 160

Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly

165 170 175

Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln

180 185 190

Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu

195 200 205

Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro

210 215 220

Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr

225 230 235 240

Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe

245 250 255

Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe

260 265 270

Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile

275 280 285

Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn

290 295 300

Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr

305 310 315 320

Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly

325 330 335

Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu

340 345 350

Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys

355 360 365

Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys

370 375 380

Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile

385 390 395 400

Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile

405 410 415

Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr

420 425 430

Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp

435 440 445

Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser

450 455 460

Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn

465 470 475 480

Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp

485 490 495

Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr

500 505 510

Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys

515 520 525

Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln

530 535 540

Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp

545 550 555 560

Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp

565 570 575

Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly

580 585 590

Trp Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser

595 600 605

Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile

610 615 620

Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu

625 630 635 640

Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro

645 650 655

Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile

660 665 670

Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys

675 680 685

Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp

690 695 700

Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr

705 710 715 720

Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr

725 730 735

Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp

740 745 750

Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile

755 760 765

Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met

770 775 780

Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn

785 790 795 800

Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr

805 810 815

Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu

820 825 830

Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile

835 840 845

Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile

850 855 860

Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly

865 870 875 880

Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys

885 890 895

Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr

900 905 910

Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val

915 920 925

Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn

930 935 940

Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser

945 950 955 960

Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile

965 970 975

Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg

980 985 990

Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr

995 1000 1005

Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser

1010 1015 1020

Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile

1025 1030 1035 1040

Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile Trp Met

1045 1050 1055

Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu

1060 1065 1070

Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp

1075 1080 1085

Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly

1090 1095 1100

Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu

1105 1110 1115 1120

Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr

1125 1130 1135

Arg Asp Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn

1140 1145 1150

Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr

1155 1160 1165

Leu Asp Phe Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys

1170 1175 1180

Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp

1185 1190 1195 1200

Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln

1205 1210 1215

Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr

1220 1225 1230

Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile

1235 1240 1245

Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu

1250 1255 1260

Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp

1265 1270 1275 1280

Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu

1285 1290

<210> 5

<211> 3843

<212> DNA

<213> Clostridium botulinum (Clostridium botulinum)

<400> 5

atgccaataa caattaacaa ctttaattat tcagatcctg ttgataataa aaatatttta 60

tatttagata ctcatttaaa tacattagct aatgagcctg aaaaagcctt tcgcattata 120

gggaatatat gggtaatacc cgatagattt tcaagagatt ctaatccaaa tttaaataaa 180

cctcctcgag ttacaagccc taaaagtggt tattatgatc ctaattattt gagtactgat 240

tctgaaaaag atacattttt aaaagaaatt ataaagttat ttaaaagaat taactctaga 300

gaaataggag aagaattaat atatagactt gcaacagaca taccctttcc tgggaataac 360

aatactccaa ttaatacttt tgattttgat gtagatttta acagtgttga tgttaaaact 420

agacaaggta acaactgggt taaaactggt agtataaatc ctagtgttat aataactgga 480

cctagagaaa acattataga cccagaaact tctacgttta aattaactaa caatactttt 540

gcggcacaag aaggatttgg tgctttatca ataatttcaa tatcacctag atttatgcta 600

acatatagta atgcaactaa taatgtagga gagggtagat tttctaagtc tgaattttgc 660

atggatccaa tactaatttt aatgcatgaa cttaatcatg caatgcataa tttatatgga 720

atagctatac caaatgatca aagaatttca tctgtaacta gtaatatttt ttattctcaa 780

tataaggtga aattagagta tgcagaaata tatgcatttg gaggtccaac tatagacctt 840

attcctaaaa gtgcaaggaa atattttgag gaaaaggcat tggattatta tagatccata 900

gctaaaagac ttaatagtat aactactgca aatccttcaa gctttaataa atatatagga 960

gaatataaac agaaacttat tagaaagtat agattcgtag tagaatcttc aggtgaagtt 1020

gcagtagatc gtaataagtt tgctgagtta tataaagaac ttacacaaat atttacagaa 1080

tttaactacg ctaaaatata taatgtacaa aataggaaaa tatatctttc aaatgtatat 1140

actccggtta cggcaaatat attagacgat aatgtttatg atatacaaaa tggatttaac 1200

atacctaaaa gtaatttaaa tgtactattt atgggtcaaa atttatctcg aaatccagca 1260

ttaagaaaag tcaatcctga aaatatgctt tatttattta caaaattttg ccataaagca 1320

atagatggta gatcattata taataaaaca ttagattgta gagagctttt agttaaaaat 1380

actgacttac cctttatagg tgatattagt gatatcaaaa ctgatatatt tttaagcaaa 1440

gatattaatg aagaaactga agttatagac tatccggaca atgtttcagt ggatcaagtt 1500

attctcagta agaatacctc agaacatgga caactagatt tattataccc tattattgaa 1560

ggtgagagtc aagtattacc gggagagaat caagtctttt atgataatag aactcaaaat 1620

gttgattatt tgaattctta ttattaccta gaatctcaaa aactaagtga taatgttgaa 1680

gattttactt ttacgacatc aattgaggaa gctttggata atagtggaaa agtatatact 1740

tactttccta aactagctga taaagtaaat acgggtgttc aaggtggttt atttttaatg 1800

tgggcaaatg atgtagttga agattttact acaaatattc taagaaaaga tacattagat 1860

aaaatatcag atgtatcagc tattattccc tatataggac ctgcattaaa tataagtaat 1920

tctgtaagaa ggggaaattt tactgaagca tttgcagtta ccggtgtaac tattttatta 1980

gaagcgtttc aagaatttac aatacctgca cttggtgcat ttgtgattta tagtaaggtt 2040

caagaaagaa acgagattat taaaactata gataattgtt tagaacaaag gattaaaaga 2100

tggaaagatt catatgaatg gatgatagga acgtggttat ccaggattac tactcaattt 2160

aataatataa gttatcaaat gtatgattct ttaaattatc aggcagatgc aatcaaagat 2220

aaaatagatt tagaatataa aaaatactca ggaagtgata aagaaaatat aaaaagtcaa 2280

gttgaaaatt taaaaaatag tttagatata aaaatctcgg aagcaatgaa taatataaat 2340

aaatttatac gagaatgttc tgtaacatac ttatttaaaa atatgctccc taaagtaatt 2400

gatgaattaa ataagtttga tttaaaaact aaaacagaat taattaatct tatagatagt 2460

cataatatta ttctagttgg tgaagtagat agattaaaag caaaagtaaa tgagagtttt 2520

gaaaatacaa taccctttaa tattttttca tatactaata attctttatt aaaagatata 2580

attaatgaat atttcaatag tattaatgat tcaaaaattt tgagcttaca aaacaaaaaa 2640

aatgctttag tggatacatc aggatataat gcagaagtga ggctagaagg tgatgttcaa 2700

gttaatacga tatatacaaa tgattttaaa ttaagtagtt caggagataa aattatagta 2760

aatttaaata ataatatttt atatagcgct atttatgaga actctagtgt tagtttttgg 2820

attaagatat ctaaagattt aactaattct cataatgaat atacaataat taatagtata 2880

aaacaaaatt ctgggtggaa attatgtatt aggaatggca atatagaatg gattttacaa 2940

gatattaata gaaagtataa aagtttaatt tttgattata gtgaatcatt aagtcataca 3000

ggatatacaa ataaatggtt ttttgttact ataactaata atataatggg gtatatgaaa 3060

ctttatataa atggagaatt aaagcagagt gaaagaattg aagatttaaa tgaggttaag 3120

ttagataaaa ccatagtatt tggaatagat gagaatatag atgagaatca gatgctttgg 3180

attagagatt ttaatatttt ttctaaagaa ttaagcaatg aagatattaa tattgtatat 3240

gagggacaaa tattaagaaa tgttattaaa gattattggg gaaatccttt gaagtttgat 3300

acagaatatt atattattaa tgataattat atagataggt atatagcacc taaaagtaat 3360

atacttgtac ttgttcagta tccagataga tctaaattat atactggaaa tcctattact 3420

attaaatcag tatctgataa gaatccttat agtagaattt taaatggaga taatataatg 3480

tttcatatgt tatataatag tgggaaatat atgataataa gagatactga tacaatatat 3540

gcaatagaag gaagagagtg ttcaaaaaat tgtgtatatg cattaaaatt acagagtaat 3600

ttaggtaatt atggtatagg tatatttagt ataaaaaata ttgtatctca aaataaatat 3660

tgtagtcaaa ttttctctag ttttatgaaa aatacaatgc ttctagcaga tatatataaa 3720

ccttggagat tttcttttga aaatgcatac acgccagttg cagtaactaa ttatgagaca 3780

aaactattat caacttcatc tttttggaaa tttatttcta gggatccagg atgggtagag 3840

taa 3843

<210> 6

<211> 1280

<212> PRT

<213> Clostridium botulinum (Clostridium botulinum)

<400> 6

Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn

1 5 10 15

Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu

20 25 30

Pro Glu Lys Ala Phe Arg Ile Ile Gly Asn Ile Trp Val Ile Pro Asp

35 40 45

Arg Phe Ser Arg Asp Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val

50 55 60

Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp

65 70 75 80

Ser Glu Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg

85 90 95

Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ala Thr

100 105 110

Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp

115 120 125

Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn

130 135 140

Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly

145 150 155 160

Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr

165 170 175

Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile

180 185 190

Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asn

195 200 205

Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile

210 215 220

Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly

225 230 235 240

Ile Ala Ile Pro Asn Asp Gln Arg Ile Ser Ser Val Thr Ser Asn Ile

245 250 255

Phe Tyr Ser Gln Tyr Lys Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala

260 265 270

Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr

275 280 285

Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu

290 295 300

Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly

305 310 315 320

Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser

325 330 335

Ser Gly Glu Val Ala Val Asp Arg Asn Lys Phe Ala Glu Leu Tyr Lys

340 345 350

Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn

355 360 365

Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr

370 375 380

Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn

385 390 395 400

Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser

405 410 415

Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu

420 425 430

Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn

435 440 445

Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro

450 455 460

Phe Ile Gly Asp Ile Ser Asp Ile Lys Thr Asp Ile Phe Leu Ser Lys

465 470 475 480

Asp Ile Asn Glu Glu Thr Glu Val Ile Asp Tyr Pro Asp Asn Val Ser

485 490 495

Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu

500 505 510

Asp Leu Leu Tyr Pro Ile Ile Glu Gly Glu Ser Gln Val Leu Pro Gly

515 520 525

Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu

530 535 540

Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu

545 550 555 560

Asp Phe Thr Phe Thr Thr Ser Ile Glu Glu Ala Leu Asp Asn Ser Gly

565 570 575

Lys Val Tyr Thr Tyr Phe Pro Lys Leu Ala Asp Lys Val Asn Thr Gly

580 585 590

Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp

595 600 605

Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp

610 615 620

Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn

625 630 635 640

Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val

645 650 655

Thr Ile Leu Leu Glu Ala Phe Gln Glu Phe Thr Ile Pro Ala Leu Gly

660 665 670

Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys

675 680 685

Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser

690 695 700

Tyr Glu Trp Met Ile Gly Thr Trp Leu Ser Arg Ile Thr Thr Gln Phe

705 710 715 720

Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Asp

725 730 735

Ala Ile Lys Asp Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser

740 745 750

Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu

755 760 765

Asp Ile Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg

770 775 780

Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile

785 790 795 800

Asp Glu Leu Asn Lys Phe Asp Leu Lys Thr Lys Thr Glu Leu Ile Asn

805 810 815

Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu

820 825 830

Lys Ala Lys Val Asn Glu Ser Phe Glu Asn Thr Ile Pro Phe Asn Ile

835 840 845

Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr

850 855 860

Phe Asn Ser Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys

865 870 875 880

Asn Ala Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Leu Glu

885 890 895

Gly Asp Val Gln Val Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser

900 905 910

Ser Ser Gly Asp Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr

915 920 925

Ser Ala Ile Tyr Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser

930 935 940

Lys Asp Leu Thr Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile

945 950 955 960

Lys Gln Asn Ser Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu

965 970 975

Trp Ile Leu Gln Asp Ile Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp

980 985 990

Tyr Ser Glu Ser Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe

995 1000 1005

Val Thr Ile Thr Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn

1010 1015 1020

Gly Glu Leu Lys Gln Ser Glu Arg Ile Glu Asp Leu Asn Glu Val Lys

1025 1030 1035 1040

Leu Asp Lys Thr Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn

1045 1050 1055

Gln Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser

1060 1065 1070

Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn Val

1075 1080 1085

Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr

1090 1095 1100

Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Lys Ser Asn

1105 1110 1115 1120

Ile Leu Val Leu Val Gln Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly

1125 1130 1135

Asn Pro Ile Thr Ile Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg

1140 1145 1150

Ile Leu Asn Gly Asp Asn Ile Met Phe His Met Leu Tyr Asn Ser Gly

1155 1160 1165

Lys Tyr Met Ile Ile Arg Asp Thr Asp Thr Ile Tyr Ala Ile Glu Gly

1170 1175 1180

Arg Glu Cys Ser Lys Asn Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn

1185 1190 1195 1200

Leu Gly Asn Tyr Gly Ile Gly Ile Phe Ser Ile Lys Asn Ile Val Ser

1205 1210 1215

Gln Asn Lys Tyr Cys Ser Gln Ile Phe Ser Ser Phe Met Lys Asn Thr

1220 1225 1230

Met Leu Leu Ala Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Glu Asn

1235 1240 1245

Ala Tyr Thr Pro Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser

1250 1255 1260

Thr Ser Ser Phe Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu

1265 1270 1275 1280

<210> 7

<211> 3858

<212> DNA

<213> Clostridium botulinum (Clostridium botulinum)

<400> 7

atgacatggc cagtaaaaga ttttaattat agtgatcctg ttaatgacaa tgatatatta 60

tatttaagaa taccacaaaa taagttaatt actacacctg taaaagcttt tatgattact 120

caaaatattt gggtaatacc agaaagattt tcatcagata ctaatccaag tttaagtaaa 180

ccgcctagac ctacttcaaa gtatcaaagt tattatgatc ctagttattt atctactgat 240

gagcaaaaag atacattttt aaaagggatt ataaaattat ttaaaagaat taatgaaaga 300

gatataggaa aaaaattaat aaattattta gtagttggtt caccttttat gggagattca 360

agtacgcctg aagatacatt tgattttaca cgtcatacta ctaatattgc agttgaaaag 420

tttgaaaatg gtagttggaa agtaacaaat attataacac caagtgtatt gatatttgga 480

ccacttccta atatattaga ctatacagca tcccttacat tgcaaggaca acaatcaaat 540

ccatcatttg aagggtttgg aacattatct atactaaaag tagcacctga atttttgtta 600

acatttagtg atgtaacatc taatcaaagt tcagctgtat taggcaaatc tatattttgt 660

atggatccag taatagcttt aatgcatgag ttaacacatt ctttgcatca attgtatgga 720

ataaatatac catctgataa aaggattcgt ccacaagtta gcgagggatt tttttctcaa 780

gatggaccca acgtacaatt tgaggaatta tacacatttg gaggatcaga tgttgaaata 840

atacctcaaa ttgaaagatt acaattaaga gaaaaagcat taggtcacta taaagatata 900

gcgaaaagac ttaataatat taataaaact attccttcta gttggagtag taatatagat 960

aaatataaaa aaatattttc tgaaaagtat aattttgata aagataatac aggaaatttt 1020

gttgtaaata ttgataaatt caatagctta tattcagact tgactaatgt tatgtcagaa 1080

gttgtttatt cttcgcaata taatgttaaa aacaggactc attatttttc aaagcattat 1140

ctacctgtat ttgcaaatat attagatgat aatatttata ctataataaa cggttttaat 1200

ttaacaacta aaggttttaa tatagaaaat tcgggtcaga atatagaaag gaatcctgca 1260

ctacaaaaac ttagttcaga aagtgtagta gatttgttta caaaagtatg tttaagatta 1320

acaagaaata gtagagatga ttcaacatgt attcaagtta aaaataatac attaccttat 1380

gtagctgata aagatagcat ttcacaagaa atatttgaaa gtcaaattat tacagatgag 1440

actaatgtag aaaattattc agataatttt tcattagatg aatctatttt agatgcaaaa 1500

gtccctacta atcctgaagc agtagatcca ctgttaccca atgttaatat ggaaccttta 1560

aatgttccag gtgaagaaga agtattttat gatgatatta ctaaagatgt tgattattta 1620

aactcttatt attatttgga agcccaaaaa ttaagtaata atgttgaaaa tattactctt 1680

acaacttcag ttgaagaagc attaggttat agcaataaga tatacacatt tttacctagc 1740

ttagctgaaa aagtgaataa aggtgttcaa gcaggtttat tcttaaattg ggcgaatgaa 1800

gtagttgagg attttactac aaatattatg aaaaaagata cattggataa aatatcagat 1860

gtatcagcca taattccata tataggacct gccttaaata taggaaattc agcattaagg 1920

ggaaacttta agcaagcatt tgcaacagct ggtgtagctt ttttgttaga aggatttcca 1980

gagtttacaa tacctgcact cggtgtattt accttttata gttctattca agaaagagag 2040

aaaattatta aaactataga aaattgttta gaacaaagag ttaagagatg gaaagattca 2100

tatcaatgga tggtatcaaa ttggttgtca agaattacta ctcgatttaa tcatataagt 2160

tatcaaatgt atgattcttt gagttatcag gcagatgcaa tcaaagctaa aatagattta 2220

gaatataaaa aatactcagg aagtgataaa gaaaatataa aaagtcaagt tgaaaattta 2280

aaaaatagtt tagatgtaaa aatctcggaa gcaatgaata atataaataa atttatacga 2340

gaatgttctg taacatactt atttaaaaat atgctcccta aagtaattga tgaattaaat 2400

aagtttgatt taaaaactaa aacagaatta attaatctta tagatagtca taatattatt 2460

ctagttggtg aagtagatag attaaaagca aaagtaaatg agagttttga aaatacaata 2520

ccctttaata ttttttcata tactaataat tctttattaa aagatatgat taatgaatat 2580

ttcaatagta ttaatgattc aaaaattttg agcttacaaa ataaaaaaaa tactttgatg 2640

gatacatcag gatataacgc agaagtgaga gtagaaggca atgttcagct taatccaata 2700

tttccatttg actttaaatt aggtagttca ggggatgata gaggtaaagt tatagtaacc 2760

cagaatgaaa atattgtata taatgctatg tatgaaagtt ttagtattag tttttggatt 2820

aggataaata aatgggtaag taatttacct ggatatacta taattgatag tgttaaaaat 2880

aactcaggtt ggagtatagg tattattagt aattttttag tgtttacttt aaaacaaaat 2940

gaaaatagtg aacaagatat aaactttagt tatgatatat caaagaatgc tgcgggatat 3000

aataaatggt tttttgtaac tattactacc aatatgatgg gaaatatgat gatttatata 3060

aatggaaaat taatagatac tataaaagtt aaagagttaa ctggaattaa ttttagcaaa 3120

actataacat ttcaaatgaa taaaattcca aatactggct taattacctc agattctgat 3180

aacatcaata tgtggataag ggatttttat atctttgcta aagaattaga tgataaagat 3240

attaatatat tatttaatag cttgcaatat actaatgttg taaaagatta ttggggaaat 3300

gatttaagat atgataaaga atattacatg attaacgtaa attatatgaa tagatatatg 3360

tctaaaaaag gcaatggaat tgtttttaat acacgtaaaa ataataatga cttcaatgaa 3420

ggatataaaa ttataataaa aagaattaga ggaaatacaa atgatactag agtacgagga 3480

gaaaatgtat tatattttaa tactacaatt gataacaaac aatatagttt aggtatgtat 3540

aaaccttcta gaaatctagg gactgattta gttccactag gtgcattgga tcaaccaatg 3600

gatgagatac gtaaatatgg ttcgtttata atacaaccat gcaatacttt tgattactat 3660

gcatcacaat tatttttgtc aagtaatgca acaacaaata ggcttggaat actatcaatt 3720

ggtagttata gtttcaaact tggagatgac tattggttta atcacgaata tttaattcct 3780

gttataaaaa tagagcatta tgcttcatta ttagaatcaa catcaactca ttgggttttt 3840

gtacctgcaa gtgaataa 3858

<210> 8

<211> 1285

<212> PRT

<213> Clostridium botulinum (Clostridium botulinum)

<400> 8

Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp

1 5 10 15

Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr

20 25 30

Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu

35 40 45

Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro

50 55 60

Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp

65 70 75 80

Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg

85 90 95

Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val

100 105 110

Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp

115 120 125

Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly

130 135 140

Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly

145 150 155 160

Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly

165 170 175

Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu

180 185 190

Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn

195 200 205

Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val

210 215 220

Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly

225 230 235 240

Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly

245 250 255

Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr

260 265 270

Phe Gly Gly Ser Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Leu Gln

275 280 285

Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu

290 295 300

Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ser Ser Asn Ile Asp

305 310 315 320

Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn

325 330 335

Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser

340 345 350

Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn

355 360 365

Val Lys Asn Arg Thr His Tyr Phe Ser Lys His Tyr Leu Pro Val Phe

370 375 380

Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Ile Asn Gly Phe Asn

385 390 395 400

Leu Thr Thr Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu

405 410 415

Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu

420 425 430

Phe Thr Lys Val Cys Leu Arg Leu Thr Arg Asn Ser Arg Asp Asp Ser

435 440 445

Thr Cys Ile Gln Val Lys Asn Asn Thr Leu Pro Tyr Val Ala Asp Lys

450 455 460

Asp Ser Ile Ser Gln Glu Ile Phe Glu Ser Gln Ile Ile Thr Asp Glu

465 470 475 480

Thr Asn Val Glu Asn Tyr Ser Asp Asn Phe Ser Leu Asp Glu Ser Ile

485 490 495

Leu Asp Ala Lys Val Pro Thr Asn Pro Glu Ala Val Asp Pro Leu Leu

500 505 510

Pro Asn Val Asn Met Glu Pro Leu Asn Val Pro Gly Glu Glu Glu Val

515 520 525

Phe Tyr Asp Asp Ile Thr Lys Asp Val Asp Tyr Leu Asn Ser Tyr Tyr

530 535 540

Tyr Leu Glu Ala Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu

545 550 555 560

Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr

565 570 575

Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly

580 585 590

Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn

595 600 605

Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala Ile

610 615 620

Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg

625 630 635 640

Gly Asn Phe Lys Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu

645 650 655

Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe

660 665 670

Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn

675 680 685

Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met

690 695 700

Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Arg Phe Asn His Ile Ser

705 710 715 720

Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala

725 730 735

Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn

740 745 750

Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile

755 760 765

Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val

770 775 780

Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn

785 790 795 800

Lys Phe Asp Leu Lys Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser

805 810 815

His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val

820 825 830

Asn Glu Ser Phe Glu Asn Thr Ile Pro Phe Asn Ile Phe Ser Tyr Thr

835 840 845

Asn Asn Ser Leu Leu Lys Asp Met Ile Asn Glu Tyr Phe Asn Ser Ile

850 855 860

Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Thr Leu Met

865 870 875 880

Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Glu Gly Asn Val Gln

885 890 895

Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly Ser Ser Gly Asp

900 905 910

Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn Ile Val Tyr Asn

915 920 925

Ala Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile Arg Ile Asn Lys

930 935 940

Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp Ser Val Lys Asn

945 950 955 960

Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe Leu Val Phe Thr

965 970 975

Leu Lys Gln Asn Glu Asn Ser Glu Gln Asp Ile Asn Phe Ser Tyr Asp

980 985 990

Ile Ser Lys Asn Ala Ala Gly Tyr Asn Lys Trp Phe Phe Val Thr Ile

995 1000 1005

Thr Thr Asn Met Met Gly Asn Met Met Ile Tyr Ile Asn Gly Lys Leu

1010 1015 1020

Ile Asp Thr Ile Lys Val Lys Glu Leu Thr Gly Ile Asn Phe Ser Lys

1025 1030 1035 1040

Thr Ile Thr Phe Gln Met Asn Lys Ile Pro Asn Thr Gly Leu Ile Thr

1045 1050 1055

Ser Asp Ser Asp Asn Ile Asn Met Trp Ile Arg Asp Phe Tyr Ile Phe

1060 1065 1070

Ala Lys Glu Leu Asp Asp Lys Asp Ile Asn Ile Leu Phe Asn Ser Leu

1075 1080 1085

Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr

1090 1095 1100

Asp Lys Glu Tyr Tyr Met Ile Asn Val Asn Tyr Met Asn Arg Tyr Met

1105 1110 1115 1120

Ser Lys Lys Gly Asn Gly Ile Val Phe Asn Thr Arg Lys Asn Asn Asn

1125 1130 1135

Asp Phe Asn Glu Gly Tyr Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn

1140 1145 1150

Thr Asn Asp Thr Arg Val Arg Gly Glu Asn Val Leu Tyr Phe Asn Thr

1155 1160 1165

Thr Ile Asp Asn Lys Gln Tyr Ser Leu Gly Met Tyr Lys Pro Ser Arg

1170 1175 1180

Asn Leu Gly Thr Asp Leu Val Pro Leu Gly Ala Leu Asp Gln Pro Met

1185 1190 1195 1200

Asp Glu Ile Arg Lys Tyr Gly Ser Phe Ile Ile Gln Pro Cys Asn Thr

1205 1210 1215

Phe Asp Tyr Tyr Ala Ser Gln Leu Phe Leu Ser Ser Asn Ala Thr Thr

1220 1225 1230

Asn Arg Leu Gly Ile Leu Ser Ile Gly Ser Tyr Ser Phe Lys Leu Gly

1235 1240 1245

Asp Asp Tyr Trp Phe Asn His Glu Tyr Leu Ile Pro Val Ile Lys Ile

1250 1255 1260

Glu His Tyr Ala Ser Leu Leu Glu Ser Thr Ser Thr His Trp Val Phe

1265 1270 1275 1280

Val Pro Ala Ser Glu

1285

<210> 9

<211> 3756

<212> DNA

<213> Clostridium botulinum (Clostridium botulinum)

<400> 9

atgccaaaaa ttaatagttt taattataat gatcctgtta atgatagaac aattttatat 60

attaaaccag gcggttgtca agaattttat aaatcattta atattatgaa aaatatttgg 120

ataattccag agagaaatgt aattggtaca accccccaag attttcatcc gcctacttca 180

ttaaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtga tgaagaaaag 240

gatagatttt taaaaatagt cacaaaaata tttaatagaa taaataataa tctttcagga 300

gggattttat tagaagaact gtcaaaagct aatccatatt tagggaatga taatactcca 360

gataatcaat tccatattgg tgatgcatca gcagttgaga ttaaattctc aaatggtagc 420

caagacatac tattacctaa tgttattata atgggagcag agcctgattt atttgaaact 480

aacagttcca atatttctct aagaaataat tatatgccaa gcaatcaccg ttttggatca 540

atagctatag taacattctc acctgaatat tcttttagat ttaatgataa ttgtatgaat 600

gaatttattc aagatcctgc tcttacatta atgcatgaat taatacattc attacatgga 660

ctatatgggg ctaaagggat tactacaaag tatactataa cacaaaaaca aaatccccta 720

ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggagg tactgattta 780

aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc tgattataaa 840

aaaatagcgt ctaaacttag caaagtacaa gtatctaatc cactacttaa tccttataaa 900

gatgtttttg aagcaaagta tggattagat aaagatgcta gcggaattta ttcggtaaat 960

ataaacaaat ttaatgatat ttttaaaaaa ttatacagct ttacggaatt tgatttacga 1020

actaaatttc aagttaaatg taggcaaact tatattggac agtataaata cttcaaactt 1080

tcaaacttgt taaatgattc tatttataat atatcagaag gctataatat aaataattta 1140

aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta gaattattac accaattaca 1200

ggtagaggac tagtaaaaaa aatcattaga ttttgtaaaa atattgtttc tgtaaaaggc 1260

ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgt ggcttccgag 1320

aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatac agtaacttca 1380

aataataatt atgaaaatga tttagatcag gttattttaa attttaatag tgaatcagca 1440

cctggacttt cagatgaaaa attaaattta actatccaaa atgatgctta tataccaaaa 1500

tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaact taatgtattt 1560

ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatct cacctcttca 1620

attgatacag cattattaga acaacctaaa atatatacat ttttttcatc agaatttatt 1680

aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggataca acaagtgtta 1740

gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgc agatatttct 1800

atagttgttc catatatagg tcttgcttta aatataggaa atgaagcaca aaaaggaaat 1860

tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttga acccgagctt 1920

ttaattccta caattttagt attcacgata aaatcttttt taggttcatc tgataataaa 1980

aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaa atggaaagaa 2040

gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatt taataaaaga 2100

aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg caattaaaac aataatagaa 2160

tctaagtata atagttatac tttagaggaa aaaaatgagc ttacaaataa atatgatatt 2220

aagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataa tatagacagg 2280

ttcttaactg aaagttctat atcctattta atgaaaataa taaatgaagt aaaaattaat 2340

aaattaagag aatatgatga gaatgtcaaa acgtatttat tgaattatat tatacaacat 2400

ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaactga taccctaaat 2460

aatagtattc cttttaagct ttcttcttat acagatgata aaattttaat ttcatatttt 2520

aataaattct ttaagagaat taaaagtagt tcagttttaa atatgagata taaaaatgat 2580

aaatacgtag atacttcagg atatgattca aatataaata ttaatggaga tgtatataaa 2640

tatccaacta ataaaaatca atttggaata tataatgata aacttagtga agttaatata 2700

tctcaaaatg attacattat atatgataat aaatataaaa attttagtat tagtttttgg 2760

gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaata cactataata 2820

aattgtatga gagataataa ttcaggatgg aaagtatctc ttaatcataa tgaaataatt 2880

tggacattcg aagataatcg aggaattaat caaaaattag catttaacta tggtaacgca 2940

aatggtattt ctgattatat aaataagtgg atttttgtaa ctataactaa tgatagatta 3000

ggagattcta aactttatat taatggaaat ttaatagatc aaaaatcaat tttaaattta 3060

ggtaatattc atgttagtga caatatatta tttaaaatag ttaattgtag ttatacaaga 3120

tatattggta ttagatattt taatattttt gataaagaat tagatgaaac agaaattcaa 3180

actttatata gcaatgaacc taatacaaat attttgaagg atttttgggg aaattatttg 3240

ctttatgaca aagaatacta tttattaaat gtgttaaaac caaataactt tattgatagg 3300

agaaaagatt ctactttaag cattaataat ataagaagca ctattctttt agctaataga 3360

ttatatagtg gaataaaagt taaaatacaa agagttaata atagtagtac taacgataat 3420

cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaac tcacttattt 3480

ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaat atcatcatct 3540

ggcaatagat ttaatcaagt agtagttatg aattcagtag gaaattgtac aatgaatttt 3600

aaaaataata atggaaataa tattgggttg ttaggtttca aggcagatac tgtcgttgct 3660

agtacttggt attatacaca tatgagagat catacaaaca gcaatggatg tttttggaac 3720

tttatttctg aagaacatgg atggcaagaa aaataa 3756

<210> 10

<211> 1251

<212> PRT

<213> Clostridium botulinum (Clostridium botulinum)

<400> 10

Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg

1 5 10 15

Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser

20 25 30

Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile

35 40 45

Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly

50 55 60

Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys

65 70 75 80

Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn

85 90 95

Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro

100 105 110

Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp

115 120 125

Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu

130 135 140

Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr

145 150 155 160

Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His

165 170 175

Arg Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe

180 185 190

Arg Phe Asn Asp Asn Cys Met Asn Glu Phe Ile Gln Asp Pro Ala Leu

195 200 205

Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala

210 215 220

Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu

225 230 235 240

Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly

245 250 255

Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr

260 265 270

Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys

275 280 285

Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu

290 295 300

Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn

305 310 315 320

Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu

325 330 335

Phe Asp Leu Arg Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile

340 345 350

Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile

355 360 365

Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe

370 375 380

Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr

385 390 395 400

Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val

405 410 415

Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly

420 425 430

Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile

435 440 445

Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr

450 455 460

Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala

465 470 475 480

Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala

485 490 495

Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His

500 505 510

Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val

515 520 525

Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala

530 535 540

Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile

545 550 555 560

Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile

565 570 575

Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr

580 585 590

Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu

595 600 605

Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala

610 615 620

Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu

625 630 635 640

Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser

645 650 655

Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys

660 665 670

Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn

675 680 685

Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met

690 695 700

Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu

705 710 715 720

Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn

725 730 735

Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser

740 745 750

Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser

755 760 765

Tyr Leu Met Lys Ile Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu

770 775 780

Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His

785 790 795 800

Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr

805 810 815

Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp

820 825 830

Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys

835 840 845

Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp

850 855 860

Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys

865 870 875 880

Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser

885 890 895

Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr

900 905 910

Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn

915 920 925

Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg

930 935 940

Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile

945 950 955 960

Trp Thr Phe Glu Asp Asn Arg Gly Ile Asn Gln Lys Leu Ala Phe Asn

965 970 975

Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe

980 985 990

Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn

995 1000 1005

Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His

1010 1015 1020

Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg

1025 1030 1035 1040

Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu

1045 1050 1055

Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu

1060 1065 1070

Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu

1075 1080 1085

Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser

1090 1095 1100

Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg

1105 1110 1115 1120

Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser

1125 1130 1135

Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe

1140 1145 1150

Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr

1155 1160 1165

Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe

1170 1175 1180

Asn Gln Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe

1185 1190 1195 1200

Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp

1205 1210 1215

Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr

1220 1225 1230

Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp

1235 1240 1245

Gln Glu Lys

1250

<210> 11

<211> 3843

<212> DNA

<213> Clostridium botulinum (Clostridium botulinum)

<400> 11

atgccagttg taataaatag ttttaattat aatgaccctg ttaatgatga gacaatttta 60

tacatgcaga aaccatatga agaaagaagt agaaaatatt ataaagcttt tgagattatg 120

cctaatgttt ggataatgcc tgagagagat acaataggaa ctaagcctga tgagtttcag 180

gtgccggatt cattaaagaa cggaagtagt gcttattatg atcctaatta tttaaccact 240

gatgctgaaa aagatagata tttaaaaaca atgataaaat tatttaatag aattaatagt 300

aatcctacag ggaaagtttt gttagaagaa gtatcaaatg ctagaccata tttaggagat 360

gatgacacgc taattaatga attccttcca gttaatgtaa ctacaagtgt taatataaaa 420

ttttcaactg atgttgaaag ttcaataata tcgaatcttc ttgtattggg agcaggacct 480

gatatattta aagcttactg tacccccctt gtaaggttta ataagtcaga taaattaatt 540

gaaccaagta atcatggttt tggatcaatt aatatcttga cattttcacc tgagtatgaa 600

catattttta atgatattag tggagggaat cataatagta cagaatcatt tattgcagat 660

cctgcaattt cactagctca tgaattgata catgcactac atggattata cggggctaag 720

gcagttactc ataaagagtc tctagtagca gagcgaggac ctcttatgat agccgaaaag 780

cccataaggc tagaagaatt tttaactttt ggaggtgagg atttaaatat cattcctagt 840

gctatgaagg aaaaaatata taacgatctt ttagctaact atgaaaaaat agctactaga 900

cttagagaag ttaatacggc tcctcctgga tatgatatta atgaatataa agattatttt 960

caatggaagt atggactaga tagaaatgca gatggaagtt atactgtgaa tagaaataaa 1020

tttaatgaaa tttataaaaa attatatagc tttacagaga ttgacttagc aaataaattt 1080

aaagtaaaat gtagaaatac ttattttatt aaatatggat ttgtaaaagt tccaaatttg 1140

ttagatgatg atatttatac tgtatcagag gggtttaata taggtaattt agcagtaaac 1200

aatcgcggac aaaatataaa tttaaatcct aaaattattg attccattcc agataaaggt 1260

ttagtggaaa agattattaa attttgtaag agcattattc ctagaaaagg tacgaagcag 1320

tcaccgtcac tatgcattag agtaaataat agggagttat tttttgtagc ttcagaaagt 1380

agctataatg aaagtgatat taatacacct aaagaaattg acgatacaac aaatctaaat 1440

aataattata gaaataattt agatgaagtt attttagatt ataatagtga gacaatacct 1500

caaatatcaa atcgaacatt aaatacactt gtacaagaca atagttatgt gccaagatat 1560

gattctaatg gaacaagtga aatagaggaa tatgatgttg ttgactttaa tgtatttttc 1620

tatttacatg cacaaaaagt accagaaggt gaaaccaata taagtttaac ttcttcaatt 1680

gatacagcat tattagaaga atccaaagta tatacatttt tttcttcaga gtttatcgat 1740

actatcaata aacctgtaaa tgcagcacta tttatagatt ggataagcaa agtaataaga 1800

gattttacca ctgaagctac acaaaaaagt actgttgata agattgcaga catatcttta 1860

attgtaccct atgtaggtct tgctttgaat atagttattg aggcagaaaa aggaaatttt 1920

gaggaggcat ttgaattatt aggagcgggt attttattag aatttgtgcc agagcttaca 1980

attcctgtaa ttttagtgtt tacgataaaa tcctatatag attcatatga gaataaaaat 2040

aaagcaatta aagcaataaa taattcatta atcgaaagag aagcaaagtg gaaagaaata 2100

tatagttgga tagtatcaaa ttggcttact agaattaata cgcaatttaa taaaagaaaa 2160

gagcaaatgt atcaggcttt acaaaatcaa gtagatgcaa taaaaacagc aatagaatat 2220

aaatataata attatacttc agatgagaaa aatagacttg aatctaaata taatatcaat 2280

aatatagaag aagaattgaa taaaaaagtt tctttagcaa tgaaaaatat agaaagattt 2340

atgacagaaa gttctatatc ttatttaatg aaattaataa atgaagccga agttggtaaa 2400

ttaaaagaat atgataaaca tgttaagagc gatttattag actatattct ctaccataaa 2460

ttaatcttag gagagcagac aaaggaatta attgatttgg tgactagtac tttgaatagt 2520

agtattccat ttgaactttc ttcatatact aatgataaaa ttctaattat atattttaat 2580

agattatata aaaaaattaa agatagttct attttagata tgcgatatga aaataataaa 2640

tttatagata tctctggata tggttcaaat ataagcatta atggaaacgt atatatttat 2700

tcaacaaata gaaatcaatt tggaatatat agtggtaggc ttagtgaagt taatatagct 2760

caaaataatg atattatata caatagtaga tatcaaaatt ttagtattag tttctgggta 2820

accattccta aacactacag acctatgaat cgtaatcggg aatacactat aataaattgt 2880

atggggaata ataattcggg atggaaaata tcacttagaa ctattagaga ttgtgaaata 2940

atttggactt tacaagatac ttccggaaat aaggaaaaat taatttttag gtatgaagaa 3000

cttgctagta tatctgatta tataaataaa tggatttttg taactattac taataataga 3060

ttaggcaatt ctagaattta catcaatgga aatttaatag ttgaaaaatc aatttcgaat 3120

ttaggtgata ttcatgttag tgataatata ttatttaaaa ttgttggttg tgatgatgaa 3180

acgtatgttg gtataagata ttttaaagtt tttaatacgg aattagataa aacagaaatt 3240

gagactttat atagtaatga gccagatcca agtatcttaa aagactattg gggaaattat 3300

ttgctatata ataaaaaata ttatttattc aatttactaa gaaaagataa gtatattact 3360

cggaattcag gcattttaaa tattaatcaa caaagaggtg ttactggagg catatctgtt 3420

tttttgaact ataaattata tgaaggagta gaagttatta taagaaaaaa tgctcctata 3480

gatatatcta atacagataa ttttgttaga aaaaacgatc tagcatacat taatgtagta 3540

gatcatggtg tagaatatcg gttatatgct gatatatcaa ttacaaaatc agagaaaata 3600

ataaaattaa taagaacatc taatccaaac gatagcttag gtcaaattat agttatggat 3660

tcaataggaa ataattgcac aatgaatttt caaaacaatg atgggagcaa tataggatta 3720

ctaggttttc attcagatga tttggttgct agtagttggt attataacca tatacgaaga 3780

aacactagca gtaatggatg cttttggagt tttatttcta aagagcatgg ttggaaagaa 3840

taa 3843

<210> 12

<211> 1280

<212> PRT

<213> Clostridium botulinum (Clostridium botulinum)

<400> 12

Met Pro Val Val Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp

1 5 10 15

Glu Thr Ile Leu Tyr Met Gln Lys Pro Tyr Glu Glu Arg Ser Arg Lys

20 25 30

Tyr Tyr Lys Ala Phe Glu Ile Met Pro Asn Val Trp Ile Met Pro Glu

35 40 45

Arg Asp Thr Ile Gly Thr Lys Pro Asp Glu Phe Gln Val Pro Asp Ser

50 55 60

Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr

65 70 75 80

Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Met Ile Lys Leu Phe Asn

85 90 95

Arg Ile Asn Ser Asn Pro Thr Gly Lys Val Leu Leu Glu Glu Val Ser

100 105 110

Asn Ala Arg Pro Tyr Leu Gly Asp Asp Asp Thr Leu Ile Asn Glu Phe

115 120 125

Leu Pro Val Asn Val Thr Thr Ser Val Asn Ile Lys Phe Ser Thr Asp

130 135 140

Val Glu Ser Ser Ile Ile Ser Asn Leu Leu Val Leu Gly Ala Gly Pro

145 150 155 160

Asp Ile Phe Lys Ala Tyr Cys Thr Pro Leu Val Arg Phe Asn Lys Ser

165 170 175

Asp Lys Leu Ile Glu Pro Ser Asn His Gly Phe Gly Ser Ile Asn Ile

180 185 190

Leu Thr Phe Ser Pro Glu Tyr Glu His Ile Phe Asn Asp Ile Ser Gly

195 200 205

Gly Asn His Asn Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser

210 215 220

Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Lys

225 230 235 240

Ala Val Thr His Lys Glu Ser Leu Val Ala Glu Arg Gly Pro Leu Met

245 250 255

Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly

260 265 270

Glu Asp Leu Asn Ile Ile Pro Ser Ala Met Lys Glu Lys Ile Tyr Asn

275 280 285

Asp Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Arg Glu Val

290 295 300

Asn Thr Ala Pro Pro Gly Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe

305 310 315 320

Gln Trp Lys Tyr Gly Leu Asp Arg Asn Ala Asp Gly Ser Tyr Thr Val

325 330 335

Asn Arg Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr

340 345 350

Glu Ile Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr

355 360 365

Phe Ile Lys Tyr Gly Phe Val Lys Val Pro Asn Leu Leu Asp Asp Asp

370 375 380

Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn

385 390 395 400

Asn Arg Gly Gln Asn Ile Asn Leu Asn Pro Lys Ile Ile Asp Ser Ile

405 410 415

Pro Asp Lys Gly Leu Val Glu Lys Ile Ile Lys Phe Cys Lys Ser Ile

420 425 430

Ile Pro Arg Lys Gly Thr Lys Gln Ser Pro Ser Leu Cys Ile Arg Val

435 440 445

Asn Asn Arg Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu

450 455 460

Ser Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn

465 470 475 480

Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser

485 490 495

Glu Thr Ile Pro Gln Ile Ser Asn Arg Thr Leu Asn Thr Leu Val Gln

500 505 510

Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile

515 520 525

Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala

530 535 540

Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile

545 550 555 560

Asp Thr Ala Leu Leu Glu Glu Ser Lys Val Tyr Thr Phe Phe Ser Ser

565 570 575

Glu Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe Ile

580 585 590

Asp Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln

595 600 605

Lys Ser Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr

610 615 620

Val Gly Leu Ala Leu Asn Ile Val Ile Glu Ala Glu Lys Gly Asn Phe

625 630 635 640

Glu Glu Ala Phe Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Val

645 650 655

Pro Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr

660 665 670

Ile Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn

675 680 685

Ser Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile

690 695 700

Val Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys

705 710 715 720

Glu Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr

725 730 735

Ala Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg

740 745 750

Leu Glu Ser Lys Tyr Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn Lys

755 760 765

Lys Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser

770 775 780

Ser Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Glu Val Gly Lys

785 790 795 800

Leu Lys Glu Tyr Asp Lys His Val Lys Ser Asp Leu Leu Asp Tyr Ile

805 810 815

Leu Tyr His Lys Leu Ile Leu Gly Glu Gln Thr Lys Glu Leu Ile Asp

820 825 830

Leu Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser

835 840 845

Tyr Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys

850 855 860

Lys Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys

865 870 875 880

Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn

885 890 895

Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Ser Gly

900 905 910

Arg Leu Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn

915 920 925

Ser Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Thr Ile Pro Lys

930 935 940

His Tyr Arg Pro Met Asn Arg Asn Arg Glu Tyr Thr Ile Ile Asn Cys

945 950 955 960

Met Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr Ile Arg

965 970 975

Asp Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr Ser Gly Asn Lys Glu

980 985 990

Lys Leu Ile Phe Arg Tyr Glu Glu Leu Ala Ser Ile Ser Asp Tyr Ile

995 1000 1005

Asn Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser

1010 1015 1020

Arg Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn

1025 1030 1035 1040

Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly

1045 1050 1055

Cys Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asn

1060 1065 1070

Thr Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn Glu Pro

1075 1080 1085

Asp Pro Ser Ile Leu Lys Asp Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn

1090 1095 1100

Lys Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr Ile Thr

1105 1110 1115 1120

Arg Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg Gly Val Thr Gly

1125 1130 1135

Gly Ile Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val

1140 1145 1150

Ile Ile Arg Lys Asn Ala Pro Ile Asp Ile Ser Asn Thr Asp Asn Phe

1155 1160 1165

Val Arg Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp His Gly Val

1170 1175 1180

Glu Tyr Arg Leu Tyr Ala Asp Ile Ser Ile Thr Lys Ser Glu Lys Ile

1185 1190 1195 1200

Ile Lys Leu Ile Arg Thr Ser Asn Pro Asn Asp Ser Leu Gly Gln Ile

1205 1210 1215

Ile Val Met Asp Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn

1220 1225 1230

Asn Asp Gly Ser Asn Ile Gly Leu Leu Gly Phe His Ser Asp Asp Leu

1235 1240 1245

Val Ala Ser Ser Trp Tyr Tyr Asn His Ile Arg Arg Asn Thr Ser Ser

1250 1255 1260

Asn Gly Cys Phe Trp Ser Phe Ile Ser Lys Glu His Gly Trp Lys Glu

1265 1270 1275 1280

<210> 13

<211> 3894

<212> DNA

<213> Clostridium botulinum (Clostridium botulinum)

<220>

<223> n is a, c, g or t

<220>

<223> n is a, c, g or t

<400> 13

atgccagtta atataaaaan ctttaattat aatgacccta ttaataatga tgacattatt 60

atgatggaac cattcaatga cccagggcca ggaacatatt ataaagcttt taggattata 120

gatcgtattt ggatagtacc agaaaggttt acttatggat ttcaacctga ccaatttaat 180

gccagtacag gagtttttag taaagatgtc tacgaatatt acgatccaac ttatttaaaa 240

accgatgctg aaaaagataa atttttaaaa acaatgatta aattatttaa tagaattaat 300

tcaaaaccat caggacagag attactggat atgatagtag atgctatacc ttatcttgga 360

aatgcatcta caccgcccga caaatttgca gcaaatgttg caaatgtatc tattaataaa 420

aaaattatcc aacctggagc tgaagatcaa ataaaaggtt taatgacaaa tttaataata 480

tttggaccag gaccagttct aagtgataat tttactgata gtatgattat gaatggccat 540

tccccaatat cagaaggatt tggtgcaaga atgatgataa gattttgtcc tagttgttta 600

aatgtattta ataatgttca ggaaaataaa gatacatcta tatttagtag acgcgcgtat 660

tttgcagatc cagctctaac gttaatgcat gaacttatac atgtgttaca tggattatat 720

ggaattaaga taagtaattt accaattact ccaaatacaa aagaattttt catgcaacat 780

agcgatcctg tacaagcaga agaactatat acattcggag gacatgatcc tagtgttata 840

agtccttcta cggatatgaa tatttataat aaagcgttac aaaattttca agatatagct 900

aataggctta atattgtttc aagtgcccaa gggagtggaa ttgatatttc cttatataaa 960

caaatatata aaaataaata tgattttgtt gaagatccta atggaaaata tagtgtagat 1020

aaggataagt ttgataaatt atataaggcc ttaatgtttg gctttactga aactaatcta 1080

gctggtgaat atggaataaa aactaggtat tcttatttta gtgaatattt gccaccgata 1140

aaaactgaaa aattgttaga caatacaatt tatactcaaa atgaaggctt taacatagct 1200

agtaaaaatc tcaaaacgga atttaatggt cagaataagg cggtaaataa agaggcttat 1260

gaagaaatca gcctagaaca tctcgttata tatagaatag caatgtgcaa gcctgtaatg 1320

tacaaaaata ccggtaaatc tgaacagtgt attattgtta ataatgagga tttatttttc 1380

atagctaata aagatagttt ttcaaaagat ttagctaaag cagaaactat agcatataat 1440

acacaaaata atactataga aaataatttt tctatagatc agttgatttt agataatgat 1500

ttaagcagtg gcatagactt accaaatgaa aacacagaac catttacaaa ttttgacgac 1560

atagatatcc ctgtgtatat taaacaatct gctttaaaaa aaatttttgt ggatggagat 1620

agcctttttg aatatttaca tgctcaaaca tttccttcta atatagaaaa tctacaacta 1680

acgaattcat taaatgatgc tttaagaaat aataataaag tctatacttt tttttctaca 1740

aaccttgttg aaaaagctaa tacagttgta ggtgcttcac tttttgtaaa ctgggtaaaa 1800

ggagtaatag atgattttac atctgaatcc acacaaaaaa gtactataga taaagtttca 1860

gatgtatcca taattattcc ctatatagga cctgctttga atgtaggaaa tgaaacagct 1920

aaagaaaatt ttaaaaatgc ttttgaaata ggtggagccg ctatcttaat ggagtttatt 1980

ccagaactta ttgtacctat agttggattt tttacattag aatcatatgt aggaaataaa 2040

gggcatatta ttatgacgat atccaatgct ttaaagaaaa gggatcaaaa atggacagat 2100

atgtatggtt tgatagtatc gcagtggctc tcaacggtta atactcaatt ttatacaata 2160

aaagaaagaa tgtacaatgc tttaaataat caatcacaag caatagaaaa aataatagaa 2220

gatcaatata atagatatag tgaagaagat aaaatgaata ttaacattga ttttaatgat 2280

atagatttta aacttaatca aagtataaat ttagcaataa acaatataga tgattttata 2340

aaccaatgtt ctatatcata tctaatgaat agaatgattc cattagctgt aaaaaagtta 2400

aaagactttg atgataatct taagagagat ttattggagt atatagatac aaatgaacta 2460

tatttacttg atgaagtaaa tattctaaaa tcaaaagtaa atagacacct aaaagacagt 2520

ataccatttg atctttcact atataccaag gacacaattt taatacaagt ttttaataat 2580

tatattagta atattagtag taatgctatt ttaagtttaa gttatagagg tgggcgttta 2640

atagattcat ctggatatgg tgcaactatg aatgtaggtt cagatgttat ctttaatgat 2700

ataggaaatg gtcaatttaa attaaataat tctgaaaata gtaatattac ggcacatcaa 2760

agtaaattcg ttgtatatga tagtatgttt gataatttta gcattaactt ttgggtaagg 2820

actcctaaat ataataataa tgatatacaa acttatcttc aaaatgagta tacaataatt 2880

agttgtataa aaaatgactc aggatggaaa gtatctatta agggaaatag aataatatgg 2940

acattaatag atgttaatgc aaaatctaaa tcaatatttt tcgaatatag tataaaagat 3000

aatatatcag attatataaa taaatggttt tccataacta ttactaatga tagattaggt 3060

aacgcaaata tttatataaa tggaagtttg aaaaaaagtg aaaaaatttt aaacttagat 3120

agaattaatt ctagtaatga tatagacttc aaattaatta attgtacaga tactactaaa 3180

tttgtttgga ttaaggattt taatattttt ggtagagaat taaatgctac agaagtatct 3240

tcactatatt ggattcaatc atctacaaat actttaaaag atttttgggg gaatccttta 3300

agatacgata cacaatacta tctgtttaat caaggtatgc aaaatatcta tataaagtat 3360

tttagtaaag cttctatggg ggaaactgca ccacgtacaa actttaataa tgcagcaata 3420

aattatcaaa atttatatct tggtttacga tttattataa aaaaagcatc aaattctcgg 3480

aatataaata atgataatat agtcagagaa ggagattata tatatcttaa tattgataat 3540

atttctgatg aatcttacag agtatatgtt ttggtgaatt ctaaagaaat tcaaactcaa 3600

ttatttttag cacccataaa tgatgatcct acgttctatg atgtactaca aataaaaaaa 3660

tattatgaaa aaacaacata taattgtcag atactttgcg aaaaagatac taaaacattt 3720

gggctgtttg gaattggtaa atttgttaaa gattatggat atgtttggga tacctatgat 3780

aattattttt gcataagtca gtggtatctc agaagaatat ctgaaaatat aaataaatta 3840

aggttgggat gtaattggca attcattccc gtggatgaag gatggacaga ataa 3894

<210> 14

<211> 1297

<212> PRT

<213> Clostridium botulinum (Clostridium botulinum)

<220>

<223> Xaa can be any naturally occurring amino acid

<220>

<223> Xaa can be any naturally occurring amino acid

<400> 14

Met Pro Val Asn Ile Lys Xaa Phe Asn Tyr Asn Asp Pro Ile Asn Asn

1 5 10 15

Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr

20 25 30

Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu

35 40 45

Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly

50 55 60

Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys

65 70 75 80

Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe

85 90 95

Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile

100 105 110

Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys

115 120 125

Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln

130 135 140

Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile

145 150 155 160

Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile

165 170 175

Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met

180 185 190

Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu

195 200 205

Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro

210 215 220

Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr

225 230 235 240

Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe

245 250 255

Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe

260 265 270

Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile

275 280 285

Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn

290 295 300

Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys

305 310 315 320

Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys

325 330 335

Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met

340 345 350

Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr

355 360 365

Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys

370 375 380

Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala

385 390 395 400

Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn

405 410 415

Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg

420 425 430

Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu

435 440 445

Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys

450 455 460

Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn

465 470 475 480

Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile

485 490 495

Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr

500 505 510

Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys

515 520 525

Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu

530 535 540

Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu

545 550 555 560

Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr

565 570 575

Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala

580 585 590

Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser

595 600 605

Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Val Ser Ile

610 615 620

Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala

625 630 635 640

Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu

645 650 655

Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr

660 665 670

Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser

675 680 685

Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu

690 695 700

Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile

705 710 715 720

Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu

725 730 735

Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met

740 745 750

Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser

755 760 765

Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser

770 775 780

Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu

785 790 795 800

Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp

805 810 815

Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys

820 825 830

Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr

835 840 845

Thr Lys Asp Thr Ile Leu Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn

850 855 860

Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu

865 870 875 880

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 Gln Phe Lys Leu Asn Asn Ser Glu

900 905 910

Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr Asp Ser

915 920 925

Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr

930 935 940

Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile

945 950 955 960

Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn

965 970 975

Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile

980 985 990

Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys

995 1000 1005

Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile

1010 1015 1020

Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn Leu Asp

1025 1030 1035 1040

Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile Asn Cys Thr

1045 1050 1055

Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg

1060 1065 1070

Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gln Ser Ser

1075 1080 1085

Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr

1090 1095 1100

Gln Tyr Tyr Leu Phe Asn Gln Gly Met Gln Asn Ile Tyr Ile Lys Tyr

1105 1110 1115 1120

Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn

1125 1130 1135

Asn Ala Ala Ile Asn Tyr Gln Asn Leu Tyr Leu Gly Leu Arg Phe Ile

1140 1145 1150

Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn Asn Asp Asn Ile Val

1155 1160 1165

Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu

1170 1175 1180

Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Glu Ile Gln Thr Gln

1185 1190 1195 1200

Leu Phe Leu Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu

1205 1210 1215

Gln Ile Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu

1220 1225 1230

Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe

1235 1240 1245

Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys

1250 1255 1260

Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn Lys Leu

1265 1270 1275 1280

Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu Gly Trp Thr

1285 1290 1295

Glu