HK1120051A - Carrier for targeting nerve cells - Google Patents

Description

Vectors for targeting nerve cells

Technical Field

The present invention relates to a transporter protein which binds to neurons with a higher or lower affinity than the neurotoxin formed by Clostridium botulinum. The transporter is suitably taken up by receptor-mediated endocytosis. The protein can be used as a transport means to transfer other chemicals (e.g., proteases) from an acidic endosomal compartment (endosomal component) into the cytosol of neurons, which proteases are unable to physiologically cross the plasma membrane to penetrate into the cytosol of neurons. The invention relates in particular to the use of a transport protein for the introduction of an inhibitor of neurotransmitter release.

Background

Nerve cells release transmitters through exocytosis. The fusion of the membrane of the intracellular vesicle with the cytoplasmic membrane is called exocytosis. During this process, the vesicle contents are simultaneously expelled into the synaptic cleft. The fusion of these two membranes is regulated by calcium, which reacts with the protein synaptotagmin. Under the combined action of other cofactors, synaptotagmin controls the state of three so-called fusion proteins: SNAP-25(25K synaptosomal-related protein), synaptobrevin 2(synaptobrevin 2) and synapsin 1A (syntaxin 1A). SNAP-25 binds only loosely to the cytoplasmic membrane, while synaptotagmin 1A and synaptobrevin 2 are integrated into the cytoplasm and/or vesicular membrane. These three proteins bind to each other to the extent that intracellular calcium concentrations increase, as do the two membranes, which are close to each other and eventually fuse together. In the case of the release of cholinergic neurons, acetylcholine, muscle contraction, perspiration and other cholinergic excitatory responses are triggered.

The above-mentioned fusion protein is a target molecule (substrate) of a Light Chain (LC) of clostridial toxin formed by clostridium botulinum (c.botulinum), clostridium butyricum (c.butyricum), clostridium baratii (c.baratii) and clostridium tetani (c.tetani).

The anaerobic gram-positive bacterium, clostridium botulinum, produces 7 different serotypes of clostridial neurotoxin. The latter are known as botulinum toxins (BoNT/A to BoNT/G). Among these, BoNT/A and BoNT/B in particular cause dysfunction of neuroparalysis in humans and animals, known as botulism. Spores of botulinum can be found in soil, but improper sterilization and sealing can also lead to growth when home-made food is stored, and many cases of botulism can be attributed to this.

BoNT/A is the most active of all known biologically active substances. Purified BoNT/A as little as 5-6pg represents a minimal lethal dose MLD (Multiple Lethal Dose). Female Swiss Webster mice weighing 18-20g each were given an intraperitoneal injection, and one unit (Engl.: unit U) of killing half of the BoNT was defined as MLD. 7 immunologically distinct BoNTs were characterized and are designated BoNT/A, B, C1, D, E, F and G and can be distinguished by neutralization experiments with serotype-specific antibodies. BoNT of different serotypes varies among affected animal species in terms of the severity and duration of the paralysis elicited. Thus, for paralysis, in the case of rats, the potency of BoNT/A is 500 times greater than that of BoNT/B. In addition, a dose of 480U/kg body weight of BoNT/B in primates has been shown to be non-toxic. Equal amounts of BoNT/A correspond to 12 times the Lethal Dose (LD) of the substance in primates. On the other hand, the duration of paralysis in the rats after BoNT/A injection was 10 times longer than after BoNT/E injection.

BoNTs are used to treat neuromuscular disorders, characterized by skeletal muscle hyperactivity, caused by pathologically overactive peripheral nerves. BoNT/A has been approved by the U.S. food and drug administration for the treatment of blepharospasm, strabismus, hyperhidrosis, wrinkles and hemifacial spasm. The remaining BoNT serotypes are significantly less effective than BoNT/a and show shorter potency durations. The clinical efficacy of peripheral intramuscular BoNT/A administration is usually evident within a week. The duration of symptom suppression by a single BoNT/a intramuscular injection is typically about 3-6 months.

Clostridial neurotoxins specifically hydrolyze proteins of different fusion tissues. BoNT/A, C1 and E break down SNAP-25, while BoNT/B, D, F, G and tetanus neurotoxin (TeNT) attack vesicle-associated membrane protein (VAMP) 2- -also known as synaptophysin 2- -. BoNT/C1 can further break down syntaxin 1A.

Clostridium bacteria release neurotoxins, which are single-chain polypeptides, all containing 1251-1315 amino acids. Endogenous proteases then cleave each of these proteins into two strands at defined positions (scission), but the two remaining strands are still linked together by disulfide bridges. Such double-stranded proteins are also known as holotoxins (holotoxins) (see, e.g., Shone et al (1985), Eur. J. biochem.151, 75-82). The two chains have different functions. The smaller fragment, the Light Chain (LC), is Zn²⁺Endoproteases are dependent, while the larger unit, the Heavy Chain (HC), represents the transport means for the light chain. Two 50kDa fragments were generated by treatment of the HC with endopeptidase (see Gimenez et al (1993), J. protein chem.12, 351-. Half of the amino terminus (H)_NFragments) are integrated into the membrane at lower pH values and enable the penetration of LC into the cytosol of the nerve cells. And the carboxy-terminal half (H)_CFragments) to complex polysialic gangliosides (polysialinogenesis), which occurs exclusively in the nerve cell membrane and no protein receptor has been identified to date (Halpern et al (1993), Curr Top Microbial Immunol 195, 221-241). The latter explains the high neuroselectivity of clostridial neurotoxins. The crystalline structure demonstrates that BoNT/A removes three domains, which can be reconciled by a three-step mechanism of action (see, Lacy et al (1998), nat. struct. biol.5, 898-902). Furthermore, these data lead to the conclusion that: within the Hc fragment two autonomous subunits (subdomains) are present, each at 25 kDa. The first evidence of the presence of two functional subdomains is by the amino terminus (H) of the Hc fragment of TeNT_CN) And the carboxy-terminal half (Hcc), the expression of whichIn recombinant form, and shows H_CC-domain, other than H_CNThe domain binds to neuronal cells (see Herreros et al (2000), biochem. J.347, 199-204). In the later stages, H of BoNT/A and B_CCA single ganglioside (ganglioside) binding site in the domain was located and characterized (see Rummelet al (2004), mol. microbiol.51, 631-643). Sites on BoNT/B and G for binding to synaptotagmins I and II of the protein receptors defined as BoNT/B and G are also defined as H of BoNT/B and G_CCDomain regions (see Rummel et al (2004), J Biol Chem 279, 30865-70). However, this document does not disclose amino acids related to the binding pocket of BoNT/B and G.

Under physiological conditions, H_CThe fragments bind to gangliosides of neurons, which are taken up inside the cell by receptor-mediated endocytosis and reach the intrinsic vesicle circulation (vesiclecularization) through an endosomal compartment. In the acidic medium of early endosomes (early endosomes), H_NThe fragments penetrate the vesicular membrane and form pores. Each substance (X) attached to the HC by a disulfide bridge will be split off from the HC by the intracellular redox system, enter the disulfide bridge and reduce it. X will eventually appear in the cytosol.

For clostridial neurotoxins, HC is the carrier of LC, which in the final step cleaves its specific substrate in the cytosol. The complex formation and dissociation cycle of the fusion protein is interrupted and thus the release of acetylcholine is inhibited. As a result, striated muscles are weak and sweat glands stop secreting. The active phase of individual BoNT serotypes varies and depends on the presence of intact LC in the cytosol. Since all neurons possess clostridial neurotoxin receptors, not only does the release of acetylcholine be affected, but also the release of substance P, norepinephrine, gamma aminobutyric acid (GABA), glycine, endorphin, and other transmitters and hormones may be affected.

Cholinergic transmission is preferentially blocked, which can be explained by the fact that HC in the periphery enters the neuron. The central synapse is protected by the blood brain barrier which cannot cover proteins.

H of BoNT/B and G in ligand receptor studies_CC-specific amino acid residues in the domain are substituted to determine and characterize the binding pocket of the protein receptor, in order to in turn alter its affinity for the protein receptor. Mutant BoNT/B and G H_CThe affinity of the fragment was determined by synaptotagmin in the glutathione-S-transferase precipitation assay (GST-pull-down assay). The HC subjected to the same mutation was then coupled to LC-B or LC-C, respectively. The titers of these constructs were analyzed by means of isolated mouse neuro-muscular preparations (Hemi-Diapthragm-Assay, semi-Diaphragm Assay, HDA). The phrenic nerve, which consists of cholinergic motor neurons and represents the most important physiological purpose of clostridial neurotoxins, will be found in this specimen. Subsequently, low-pressure region H of BoNT/A_CCSingle amino acids in the domain were replaced, approximately localized to the protein receptor binding pocket sites of BoNT/B and G. Full-length BoNT/a single mutants were also analyzed for their modified binding capacity by HAD, and suggested modified ligand-protein receptor interactions.

In more recent years practice, the BoNT/a complex, also known as protoxin a, has been used to treat motor dystonia, as well as to reduce excessive sympathetic activity (see Benecke et al (1995), akt. neurol.22, 209ff), and to alleviate pain and migraine (see Sycha et al (2004), j. neurol.251, 19-30). The complex is composed of neurotoxin, a plurality of hemagglutinins, and a non-toxic, non-hemagglutinating protein. This complex separates within minutes at physiological pH. The resulting neurotoxin is the only component of the complex and is therapeutically relevant and can alleviate symptoms. Since deep neurological diseases are not treated, this complex needs to be injected every 3 to 4 months. Depending on the amount of heterologous protein injected, some patients produced specific BoNT/a-antibodies. These patients become resistant to the neurotoxin. Once antigen-sensitive cells recognize the neurotoxin and form antibodies, the relevant memory cells can be stored for several years. For this reason, it is important to treat patients with formulations as active as possible, in as small a dose as possible. Furthermore, this preparation does not contain any other proteins of bacterial origin, since they can act as immunological adjuvants. Such substances will attract macrophages, which will recognize both immunoadjuvants and neurotoxins, deliver them to lymphocytes and then respond by producing immunoglobulin production. Thus, only extremely pure products that do not contain any foreign proteins can be used for therapy. The resistance of patients to neurotoxins is, on a molecular level, mainly based on the presence of neutralizing antibodies.

Disclosure of Invention

In the following, the present invention provides a transporter protein (Trapo) which can overcome the above-mentioned problems in the methods known so far.

This object is achieved by a novel transport protein which can be obtained by modifying the heavy chain of the neurotoxin produced by Clostridium botulinum. Wherein

(i) The protein binds to nerve cells with higher or lower affinity than the native neurotoxin;

(ii) the protein has enhanced or reduced neurotoxicity, preferably determined by semidiaphragmatic analysis, compared to the native neurotoxin; and/or

(iii) The protein exhibits lower affinity to neutralizing antibodies than native neurotoxin.

According to a preferred embodiment of the present invention, there is provided a transporter protein that binds to nerve cells with higher or lower affinity than the native neurotoxin produced by botulinum.

According to another preferred embodiment of the present invention, there is provided a transporter protein obtained by modifying botulinum neurotoxin HC which binds specifically to nerve cells with a stronger or weaker affinity than the native neurotoxin. The transporter is suitably taken up by these cells by endocytosis.

In addition, according to a preferred embodiment of the present invention, there is provided a transporter protein obtained by modifying HC of a neurotoxin produced by Clostridium botulinum, the binding of which to a neutralizing antibody is no longer facilitated by substituting the amino acids exposed at the surface thereof, particularly at the ganglioside binding pocket and the protein receptor binding pocket.

In the following, terms are defined to facilitate understanding of the contents of the present application.

"binds to nerve cells with greater or lesser affinity than the native neurotoxin". In the present case, native neurotoxin preferably refers to a neurotoxin produced by botulinum bacterium. The native neurotoxin herein is preferably botulinum neurotoxin A and/or botulinum neurotoxin B and/or botulinum neurotoxin G obtained from botulinum. Coli, which contains in particular the same amino acid sequence as the native botulinum neurotoxin, acts in the same pharmacological manner on the native botulinum neurotoxin and is referred to as recombinant botulinum neurotoxin wild type. The neural cell referred to in this case means a cholinergic motor neuron. Preferably, the transporter specifically binds to a molecule associated with the cytoplasmic membrane, a transmembrane protein, a synaptophysin, a synaptotacin family or a protein of synaptotacin glycoprotein 2(SV2), preferably synaptotacin I and/or synaptotacin II and/or SV2A, SV2B or SV2C, particularly preferably human synaptotacin I and/or human synaptotacin II and/or human SV2A, SV2B or SV 2C. The binding is preferably determined in vitro, particularly preferably by using the GST-pull-down assay as set forth in detail in the examples.

"the protein has enhanced or reduced neurotoxicity compared to the native neurotoxin". In the present case, native neurotoxin refers to the neurotoxin produced by botulinum toxin. Native neurotoxin in this context preferably means botulinum neurotoxin A and/or botulinum neurotoxin B and/or botulinum neurotoxin G obtained from botulinum. Coli, which contains in particular the same amino acid sequence as the native botulinum neurotoxin, acts in the same pharmacological manner on the native botulinum neurotoxin and is referred to as recombinant botulinum neurotoxin wild type. The neural cell referred to in this case means a cholinergic motor neuron. Neurotoxicity is preferably determined by means of a hemiseptal assay (HDA) known in the art. The neurotoxicity of the mutants is preferably determined by the method described by Habermann et al (cf. Habermann et al, Nauyn Schmiedeberg's Arch. Pharmacol.311(1980) 33-40).

"neutralizing antibody". Neutralizing antibodies against botulinum neurotoxin are known: (H, Wohlfarth K, Frevert J, Dengler R, Bigalke H. neutrallizing and nonneutralizationnucleotides, therpeutic sequences, exp. neuron.1997 Sep; 147(1): 96-102). It has been found that antibodies which neutralize neurotoxins are particularly active against neurotoxins such as neurotoxin H_CC-ganglioside binding pockets within the domain interact with protein receptor binding pockets. If the surrounding surface of the binding pocket in the neurotoxin is modified by amino acid substitutions while not negatively impairing the neurotoxin function, the neutralizing antibodies lose their binding sites and the mutated neurotoxin is no longer neutralized.

The term "modification of the heavy chain of the neurotoxin produced by botulinum bacterium". The amino acid and/or nucleic acid sequences of the botulinum-produced neurotoxin Heavy Chain (HC) are typically purchased from publicly accessible databases for each of the known serotypes a to G (see also table 1). Modifications herein refer to: deletion, addition, or insertion of at least one amino acid into the amino acid sequence, or substitution of at least one amino acid in the native neurotoxin with another amino acid, either native or non-native, and/or post-translational modification of an amino acid in a defined amino acid sequence. Post-translational modifications herein include glycosylation, acetylation, acylation, deamination, phosphorylation, prenylation, glycosylphosphatidyl myoxylation, and further modifications well known to those skilled in the art.

The heavy chain of the botulinum neurotoxin comprises 3 subdomains, namely a translocation domain H of 50kDa at the amino terminus_NFollowed by 25kDa size H_CN-domain, and H of 25kDa in size at the carboxy terminus_CC-a domain. H_CN-and H_CCThe domains are denoted together as H_C-a fragment. From table 1 it is evident that the amino acid portion of the respective subdomains of the individual serotypes and their variations are evident.

"ganglioside receptors"

The heavy chain HCs of botulinum neurotoxin exhibit a high affinity for peripheral nerve cells, mainly modulated by interaction with polysialic acid ganglioside complexes-glycolipids consisting of more than one sialic acid (see Halpern et al (1995), curr. top. microbiol. immunol.195, 221-41; WO 2006/02707). Light chain LCs that bind to them are therefore only able to reach this type of cell and are only active in these cells. BoNT/A and B bind only one ganglioside molecule, GT 1B.

For BoNT/B and BoNT/G, the protein receptors are synaptotagmin I and synaptotagmin II. For BoNT/A, the protein receptor is synaptic vesicle glycoprotein 2(SV2), preferably SV2A, SV2B and SV 2C.

It has now been found that 13 isoforms belong to the synaptotagmin family. They are characterized in that they both have two carboxyl terminal Ca groups capable of binding²⁺C2-knotA domain, a central transmembrane domain (TMD) that anchors synaptotagmin in the synaptic vesicle capsule, and an amino terminus of varying length. In Ca²⁺After ion influx, the fusion of synaptic vesicles with the cytoplasmic membrane is initiated, and the luminal amino terminus of the synaptotagmin is revealed extracellularly and may become the receptor anchor for BoNT/B and G. Similarly, the fourth luminal domain of the SV2 isoform may interact extracellularly with BoNT/A after exocytosis.

The single amino acid mutations of the binding pocket are characterized by being modified by specific mutations to make binding to the protein receptor difficult or inhibited. For this purpose, H of BoNT/B and BoNT/G is expressed in E.coli_CFragments and isolated in recombinant form in putative binding pockets as wild type or substitution reactions with single amino acids (mutant/substituted). For the in vitro study of the interaction between BoNT/B and BoNT/G, and between synaptotagmin I and synaptotagmin II, in the GST-pull-down assay the respective GST-synaptotagmin-fusion proteins were respectively combined with different amounts of the respective BoNT/B or H of BoNT/G_CThe fragments are incubated and phase separated. Free H_CFragment remaining in the supernatant after isolation, while bound BoNT H_CThe fragment together with the GST-synaptotagmin-fusion protein can be detected in a solid phase. Each H is_CGST-pull-down tests show identical results after the fragments have been replaced with full-length BoNT/B and BoNT/G, respectively.

It was found herein that wild-type BoNT/B binds when both ganglioside complexes and synaptotagmin I with transmembrane protein domains are present; and synaptic binding protein II binds, regardless of whether a transmembrane domain and ganglioside complex are present. It is possible to significantly enhance or attenuate the interaction of BoNT/B with two synaptotagmin molecules by specifically substituting amino acids at its receptor binding site (FIG. 1).

In addition, studies have shown that for the binding of wild-type BoNT/G to synaptotagmins I and II, binding occurs for each case regardless of whether the synaptotagmins I and II have transmembrane domains, and whether a ganglioside complex is present. By making specific amino acid substitutions similar to BoNT/B in the protein receptor binding site of BoNT/G, it may be possible to significantly enhance or attenuate its interaction with both synaptotagmin molecules I and II (FIG. 2).

The titers of the full-length versions of wild-type BoNT/A, B and G were determined by HAD and dose-effect plots were plotted (fig. 3 and 6). The titers of the different full-length forms of BoNT/A, B and G single mutants were subsequently also determined by HAD (fig. 6) and plotted against the titer of wild-type BoNT/B and G by the titer function employed (fig. 4 and 5). For example, substitution of valine at position 1118 with aspartic acid or lysine at position 1192 with glutamic acid in BoNT/B resulted in a reduction of the potency to < 2%. In contrast, mutations in which tyrosine at position 1183 was replaced with leucine or arginine, respectively, resulted in a significant increase in BoNT/B titers (fig. 4). Modification of tyrosine at position 1256 to phenylalanine likewise resulted in increased titers, while mutations in glutamic acid at position 1200 substituted with glutamine, lysine or tyrosine resulted in a considerable reduction in the titer of BoNT/G (fig. 5). For BoNT/A, modification of serine to arginine or tyrosine at position 1207 increased the potency, while the substitution of lysine to glutamic acid at position 1260 resulted in a dramatic decrease in BoNT/A potency (FIG. 6).

According to a preferred embodiment, the transporter exhibits an affinity which is at least 15% higher or at least 15% lower than the native neurotoxin. The transporter protein may preferably exhibit an affinity which is at least 50% higher or lower, particularly preferably at least 80% higher or lower, and particularly preferably at least 90% higher or lower, than the native neurotoxin.

According to a preferred embodiment, the modification of HC occurs at H of a given neurotoxin_C-a fragment region. If the modification comprises a substitution, deletion, insertion or addition, the latter may be performed by methods herein known to those skilled in the art, for example, by specific gene mutation methods. The modifications to the amino acids in the native neurotoxin herein can use amino acids that are natural or unnatural. In principle amino acids are divided into different physicochemical groups. Aspartic acid and glutamic acid belong to negatively charged amino acids. Histidine, arginine and lysine belong to positively charged amino acids. Asparagine, glutamine, serine, threonine, cysteine and tyrosine belong to the polar amino acids. Glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine and tryptophan belong to the nonpolar amino acids. Histidine, phenylalanine, tyrosine and tryptophan contain aromatic side chain groups. It is generally preferred to substitute an amino acid with an amino acid belonging to another physicochemical group.

According to a preferred embodiment of the invention, the protransporters are botulinum neurotoxin serotypes A to G. The amino acid sequence of the natural neurotoxin herein can be obtained from publicly available databases as follows:

table 1: amino acid sequence database numbering and subdomain distribution of seven botulinum neurotoxins

H for these botulinum neurotoxins_CFragments, preferably modified amino acids at amino acid positions in the following proteins:

positions 867 to 1296 of botulinum neurotoxin serotype A,

positions 866 to 1291 of botulinum neurotoxin serotype B,

positions 864 to 1291 or 1280 of botulinum neurotoxin serotype C1,

positions 860 to 1276 or 1285 of botulinum neurotoxin serotype D,

position 843 to 1251 or position 1252 of botulinum or Clostridium butyricum (C.butyricum) neurotoxin serotype E,

botulinum or Clostridium barati (C. baratii) neurotoxin serotype F861 to 1274, 862 to 1280 or 1278 and 854 to 1268,

positions 861 to 1297 of botulinum neurotoxin serotype G.

Thus, at least one amino acid in the above positions is preferably post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or unnatural amino acid.

According to a preferred embodiment, the neurotoxin is botulinum neurotoxin serotype a. In this example, it is preferred that at least one amino acid at the following positions in botulinum neurotoxin serotype a is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or unnatural amino acid: threonine at position 1195, arginine at position 1196, glutamic acid at position 1199, lysine at position 1204, isoleucine at position 1205, leucine at position 1206, serine at position 1207, leucine at position 1209, aspartic acid at position 1213, leucine at position 1217, phenylalanine at position 1255, arginine at position 1256, isoleucine at position 1258 and/or lysine at position 1260. It is particularly preferred to carry out the above-described modification of the arginine at position 1196, the glutamic acid at position 1199, the serine at position 1207, the phenylalanine at position 1255, the arginine at position 1256, the isoleucine at position 1258 and/or the lysine at position 1260 in the sequence of botulinum neurotoxin serotype A protein. In particular, it is preferable to replace serine at position 1207 with arginine or tyrosine, and to replace lysine at position 1260 with glutamic acid.

According to a preferred embodiment, the neurotoxin is botulinum neurotoxin serotype B. In this case, it is preferred that at least one amino acid at the following positions in botulinum neurotoxin serotype B is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or unnatural amino acid: lysine at position 1113, aspartic acid at position 1114, serine at position 1116, proline at position 1117, valine at position 1118, threonine at position 1182, tyrosine at position 1183, phenylalanine at position 1186, lysine at position 1188, glutamic acid at position 1191, lysine at position 1192, leucine at position 1193, phenylalanine at position 1194, phenylalanine at position 1204, phenylalanine at position 1243, glutamic acid at position 1245, lysine at position 1254, aspartic acid at position 1255 and tryptophan at position 1256. It is particularly preferred to carry out the above-described modifications of valine at position 1118, tyrosine at position 1183, glutamic acid at position 1191, lysine at position 1192, glutamic acid at position 1245 and tryptophan at position 1256 in the botulinum neurotoxin serotype B protein sequence. In particular, it is preferable to replace tyrosine at position 1183 and glutamic acid at position 1191 with leucine.

According to another preferred embodiment, the neurotoxin is botulinum neurotoxin serotype G. In this example, it is preferred that at least one amino acid at the following positions in botulinum neurotoxin serotype G is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or unnatural amino acid: phenylalanine at position 1121, lysine at position 1123, alanine at position 1124, serine at position 1125, methionine at position 1126, valine at position 1190, leucine at position 1191, serine at position 1194, glutamic acid at position 1196, threonine at position 1199, glutamic acid at position 1200, leucine at position 1201, phenylalanine at position 1202, phenylalanine at position 1212, phenylalanine at position 1248, lysine at position 1250, asparagine at position 1251, and tyrosine at position 1262. It is particularly preferred to carry out the above-described modifications of methionine at position 1126, leucine at position 1191, threonine at position 1199, glutamic acid at position 1200, lysine at position 1250 and tyrosine at position 1262 in the sequence of botulinum neurotoxin serotype G protein. In particular, tyrosine at position 1262 is preferably substituted with phenylalanine.

The transporters provided herein exhibit increased or decreased affinity of their protein binding domains, particularly for molecules belonging to the synaptotagmin family or molecules belonging to synaptoblematic vesicle glycoprotein 2.

Another embodiment of the invention relates to a composition comprising a transporter according to the invention and at least one intervening molecule (X). The intervening molecule may be a small organic molecule, a peptide or a protein; preferably, the transport protein is covalently bound via a peptide, ester, ether, sulfide, disulfide or carbon-carbon bond.

In addition, such intervening molecules include all known therapeutically active substances. Cytostatic agents, antibiotics, antiviral agents, but immunoglobulins are preferred herein.

In a preferred embodiment, the transport protein is a protease that cleaves one or more neurotransmitter-releasing tissue proteins, the protease being selected from the group of neurotoxins consisting of LC of botulinum neurotoxin, in particular serotypes A, B, C1, D, E, F and G or a proteolytically active fragment of LC of botulinum neurotoxin. In particular of serotypes A, B, C1, D, E, F and G, which fragment exhibits a proteolytic activity of at least 0.01% of the native protease, preferably at least 5%, particularly preferably at least 50%, in particular at least 90%. Preferably the transporter and the protease are from the same serotype of botulinum neurotoxin, especially preferably the H of the transporter_NA domain, and a protease derived from botulinum neurotoxin serotype a. The sequence of the protease can be obtained from an accessible database, the database numbers of which are shown in table 1. The proteolytic activity of the protease is carried out by means of substrate separation kineticsAssays (see Binz et al (2002), Biochemistry 41(6), 1717-23).

According to another embodiment of the present invention, a process for producing a transporter protein is provided. In the present case, the first step provides a nucleic acid encoding a transporter. The encoding nucleic acid herein refers to RNA, DNA or a mixture thereof. In view of its nuclease resistance, the nucleic acid may be further modified, for example by insertion of a phosphorothioate (phosphorothioate) linkage. The nucleic acid can be produced from a starting nucleic acid (starting nucleic acid), which can be obtained, for example, from a genomic or cDNA clone-database. Alternatively, the nucleic acid can be produced directly by solid phase synthesis. Suitable methods are well known to those skilled in the art. It is assumed that specific modifications of the starting nucleic acids can be made, for example by site-directed specific mutagenesis, resulting in additions, insertions, deletions and/or substitutions at least at the amino acid level. The nucleic acid is then operably linked to an appropriate promoter. Suitable promoters for expression in known expression systems are well known to those skilled in the art. In this case, the choice of promoter depends on the expression system used for expression. Generally, a constitutive promoter (constitutive promoter) is preferred, but an inducible promoter (inducible promoter) may also be used. Constructs produced in such a way include: at least one partial vector, in particular a regulatory element; a vector selected from the group consisting of lambda-derivatives, adenoviruses, baculoviruses, vaccinia viruses, simian monkey nephrovirus 40(SV40-viruses), and retroviruses. Preferably, the vector is capable of expressing the nucleic acid in a given host cell.

The invention further provides host cells comprising the vectors and suitable for expressing the vectors. Many prokaryotic and eukaryotic expression systems of choice are known in the art, for example host cells selected from e.coli or bacillus subtilis in prokaryotic cells, saccharomyces cerevisiae and pichia pastoris in eukaryotic cells. Preferably, the host cell is non-glycosylated like botulinum, although higher eukaryotic cells, such as insect cells or mammalian cells, may also be used.

According to a preferred embodiment, the nucleic acid encodes H of a botulinum neurotoxin_C-a fragment. The nucleic acid comprises an endonuclease interface flanked by nucleic acids encoding Hc fragments, which endonuclease sites coincide with other Hc fragment sites of botulinum neurotoxin, in order to facilitate modular replacement of these in genes encoding transporters, while maintaining amino acid sequence similarity.

If a composition is provided according to the invention which, in addition to the transport system, further comprises at least one intervening molecule and the intervening molecule, peptide or protein is functionalized with a carboxy-terminal cysteine or thiol group, the peptide and/or protein may then be produced recombinantly, for example using a binary vector or a plurality of host cells, in a manner analogous to that described above. If the same host cell is used for both expression of the transporter and the peptide or protein, it is preferred that the intermolecular disulfide bond is formed in situ. For more efficient production in the same host cell, the nucleic acid encoding the peptide or protein may also be translated in the same reading frame with the nucleic acid encoding the transporter protein, resulting in the production of a single-chain polypeptide. In this case it is preferred that the intramolecular disulphide bonds are formed in situ. In order to carry out simple hydrolysis of the peptide cross-links which likewise occur between transporter protein and peptide and/or protein, an amino acid sequence is inserted at the amino terminus of the transporter protein which is specifically recognized and cleaved by the protease thrombin or by a specific host cell endoprotease.

Surprisingly, if the sequence CXXXZKTKSLVPRGSKBXXC (SEQ ID NO: 1) is inserted, wherein X represents any desired amino acid and Z and B are each independently selected from alanine, valine, serine, threonine and glycine, it is effectively cleaved in vivo by an endogenous protease of a bacterial host, particularly E.coli. The insertion of this insertion sequence between the transporter and the peptide or protein can provide advantages for a later stage of post-treatment, for example, the use of thrombin treatment is no longer required. Coli K12 is particularly preferred.

Preferably, the insertion sequence forms a loop having 18 to 20 amino acids.

If this is not feasible, after separate purification of the transporter and protein, appropriate intermolecular disulfide bonds may be formed between the transporter and protein by subsequent oxidation processes well known to those skilled in the art. Peptides or proteins may also be obtained directly by synthesis or fragment condensation, suitable methods being well known to those skilled in the art.

The transporter protein and the peptide or protein are then purified separately. For this purpose, methods known to the person skilled in the art can be used, for example ion chromatography or electrophoresis.

Another embodiment in the art relates to a pharmaceutical composition comprising the transporter or a composition, and any pharmacologically acceptable excipients, diluents and/or additives.

The pharmaceutical composition is suitable for oral, intravenous, subcutaneous, intramuscular and topical administration. Intramuscular administration is preferred herein. One dosage unit of the pharmaceutical composition comprises about 0.1pg to 1mg of a transporter and/or a composition according to the invention.

The pharmaceutical composition is suitable for the treatment of neurotransmitter dysfunction and dysfunction such as the following: hemifacial spasm, spasmodic torticollis, blepharospasm, spasticity, dystonia, migraine, pain, cervical and lumbar spine dysfunction, strabismus, salivation, wound healing, snoring and depression. Cervical and lumbar spine dysfunction, strabismus, hypersalivation and depressive illness.

Another embodiment of the present invention comprises a cosmetic composition comprising a transporter protein and cosmetically acceptable excipients, diluents and/or additives. The cosmetic composition is suitable for treating hyperhidrosis and facial wrinkles.

Drawings

FIG. 1: h for wild type and mutant BoNT/B in the Presence or absence of gangliosides by GST-pull-down assay_CIn vitro studies of the binding of the fragment to the shortened GST-syt I and GST-syt II fusion proteins.

FIG. 2: h for wild type and mutant BoNT/G in the Presence or absence of gangliosides by GST-pull-down assay_CIn vitro studies of the binding of the fragment to the shortened GST-syt I and GST-syt II fusion proteins.

FIG. 3: dose-response relationship of BoNT/B and G wild-type in HDA. The duration of paralysis was compared between the single mutant and the related wild type using the titer function.

FIG. 4: increased and decreased neurotoxicity of the single mutants compared to BoNT/B wild-type in HDA.

FIG. 5: increased and decreased neurotoxicity of the single mutants compared to BoNT/G wild-type in HDA.

FIG. 6: graph of dose-response relationship between BoNT/B wild type and BoNT/A single mutant in HDA.

Detailed Description

In particular, the invention comprises a transporter protein (Trapo) formed by modifying the HC of a neurotoxin produced by clostridium botulinum, preferably binds specifically to a neuron, is preferentially taken up intracellularly by receptor-mediated endocytosis, and is translocated from the acidic endosomal compartment into the cytosol of the neuron. The protein is used as a transport means for introducing into cells proteases and other substances which bind to the transport means and which cannot physiologically penetrate the cell membrane and reach the cytosol of nerve cells. The substrates of the proteases are proteins and polypeptides localized in the cell and involved in neurotransmitter release. Upon substrate separation, specific functions of neurons are blocked; but the cells themselves are not damaged. One of these functions is exocytosis, which causes neurotransmitter release. If transmitter release is inhibited, signaling from cell to cell is blocked. For example, if acetylcholine release at the neuromuscular contact point is inhibited, striated muscles will be paralyzed. This effect can be used clinically if the transporter is applied to the nerve terminal of spastic or dystonic muscles. Other active substances are, for example, substances which exhibit an antiviral action. In conjunction with transporters, they can be used to treat viral infections of the nervous system. The invention also relates to the use of a transporter protein for inhibiting neurotransmitter release.

Transporters with relatively low affinity can bind to nerve cells, but are not absorbed by them. These transporters are therefore suitable as a specific transport means against the surface of nerve cells.

If patients are treated with botulinum protoxins A and B, injection of these non-human proteins can result in the formation of antibodies despite the low doses used, rendering the treatment ineffective and thus necessitating cessation of use to prevent anaphylactic shock. By administering substances with the same mechanism of action but higher transport efficiency of the enzyme activity, the dosage can be significantly reduced and no antibodies are produced. These properties are attributed to the transporters described herein.

Although application examples are given, the appropriate mode of application and dosage are usually determined separately by the attending physician. Such decisions should conventionally be made by a physician versed in the relevant field of expertise. Thus, for example, the mode of application and neurotoxin dosage can be selected as described herein according to the invention based on criteria such as the solubility of the neurotoxin chosen or the intensity of the pain sensation to be treated.

The interval between treatments with native protoxins a and B from botulinum bacteria typically averages 3 to 4 months. Extending this interval will reduce the risk of antibody formation and allow longer treatment cycles for BoNT. An increase in LC in the cytosol will delay its breakdown with time and thus prolong the duration of action. The transporters described herein show higher affinity and absorption rate than native HC.

The following examples are given only for illustrating the present invention and are not to be construed as a limiting manner thereof.

Materials and methods

Preparation of recombinant protein and construction of plasmid thereof

Using appropriate primers, chromosomal DNAs encoding BoNT/A (AAA23262), BoNT/B (AAA23211), and BoNT/G (CAA52275) and an expression vector pQe3(Quiagen AG) as starting vectors, a PCR method was used to obtain a desired plasmid for expressing recombinant H of BoNT/B in E.coli_CFragment, recombinant H of BoNT/G_CFragments, and recombinant H of full-length forms BoNT/A, B and G with carboxy-terminal StrepTag_C-fragments, wherein StrepTag is used for affinity purification. Shortened variants of murine-synaptotagmin I (syt I) (1-53 amino acids; 1-82 amino acids) and murine-synaptotagmin II (syt II) (1-61 amino acids; 1-90 amino acids) were cloned into the GST-encoded vector pGEX-2T (Amersham Biosciences AB). The nucleic acid sequences of all plasmids were confirmed by DNA sequencing. Recombinant H_CFragments and H of full-length form BoNT_CColi-strain M15[ pRep4 at room temperature]Was induced for 10 hours and purified on a Streptactin-matrix (IBA GmbH) according to the manufacturer's instructions. GST-fusion protein from e.coli BL21 was isolated by using glutathione immobilized on agarose gel particles. Fractions containing the desired protein fragment were pooled and dialyzed against Tris-NaCl-Triton buffer (20mM Tris-HCl, 150mM NaCl, 0, 5% Triton X-100, pH 7, 2).

GST-pull-down

Adding or not adding bovine brain spirit in a Tris-NaCl-triton-buffer system with the total volume of 180 mu lGST fusion proteins (0.12 nmol each) immobilized on 10. mu.l GT-lipoglycogel microparticles were reacted with H at 4 ℃ in the presence of a transganglioside mixture (18% GM1, 55% GD1a, 10% GT1b, 2% other gangliosides, Calbiochem; 20. mu.g each)_C-fragments (0.1nmol) incubated for 2 h. The microparticles were centrifuged and collected, the supernatant removed, and each isolated microparticle was washed 3 times with 400. mu.l of the same buffer. The pellet fraction after rinsing was boiled in SDS-sample buffer and studied together with the supernatant fraction by SDS-PAGE and Coomassie blue staining.

BoNT/B wild-type binds only in the presence of ganglioside complexes and synaptotagmin I with a transmembrane domain, and binds in synaptotagmin II with or without a transmembrane domain. By specifically substituting amino acids at the protein receptor binding site of BoNT/B, its interaction with both synaptotagmin molecules can be significantly enhanced (E1191L; Y1183L) or attenuated (V1118D; K1192E) (FIG. 1).

For BoNT/G wild-type, studies have shown that it binds to synaptotagmins I and II, regardless of whether both of these synaptotagmins carry transmembrane domains, and regardless of whether transganglioside complexes are present. By specifically substituting amino acids located at the protein receptor binding site of BoNT/G similarly to BoNT/B, the interaction between BoNT/G and these two synaptotagmin molecules can be significantly enhanced (Y1262F) or attenuated (Q1200E) (FIG. 2).

H for recombinant BoNT/B and G_CDetection of binding between the fragment and the isolated, immobilized ganglioside it was found that by introducing the mutant into the syt-binding pocket, impairment of the function of the adjacent ganglioside binding pocket could be excluded and H was obtained_C-an adequate conclusion of the complete three-dimensional structure of the fragment. These results give mutant H which also exhibits BoNT/B and G_CSupport of CD spectroscopic studies and thermal denaturation experiments of the three-dimensional structure of the fragments.

MouseSemi-diaphragm analysis (HDA)

The neurotoxicity of BoNT/A, B and G mutants was determined by the method described by Habermann et al (Habermann et al, Nauyn Schmiedeberg's Arch. Pharmacol.311(1980), 33-40).

The titers of the full-length form of BoNT/A, B and the G wild type, as determined in HDA, are shown by dose-effect graphs (FIGS. 3 and 6). Titers of the different full-length forms BoNT/A, B and G single mutants were subsequently also determined by HAD (fig. 6), and full-length forms BoNT/A, B and G wild-type were compared by mapping using a titer function (fig. 4 and 5). Substitution of valine at position 1118 to aspartic acid or lysine at position 1192 to glutamic acid in BoNT/B significantly reduced the potency to < 2%. In contrast, mutation of tyrosine 1183 to leucine or arginine, respectively, resulted in a significant increase in BoNT/B titers (fig. 4). Modification of tyrosine to phenylalanine at position 1256 also increased the potency of BoNT/G; whereas mutation of glutamic acid to glutamine, lysine or tyrosine at position 1200 resulted in a significant reduction in the potency of BoNT/G (FIG. 5). For BoNT/A, modification of serine to arginine or tyrosine at position 1207 increased the potency, while mutation of lysine to glutamic acid at position 1260 resulted in a strong attenuation of the BoNT/A potency (FIG. 6).

Sequence listing

<110> Toxogene Co., Ltd

McLee pharmaceutical Co Ltd

<120> vector for targeting nerve cells

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<170>PatentIn version 3.3

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<213> Artificial sequence

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<223> X can be any natural amino acid

<220>

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<222>(5)..(5)

<223> X may be selected from alanine, valine, serine, threonine and glycine

<220>

<221>MISC_FEATURE

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<223> X may be selected from alanine, valine, serine, threonine and glycine

<220>

<221>MISC_FEATURE

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<223> X can be any natural amino acid

<400>1

Cys Xaa Xaa Xaa Xaa Lys Thr Lys Ser Leu Val Pro Arg Gly Ser Lys

1 5 10 15

Xaa Xaa Xaa Cys

20

Claims (45)

1. A transporter protein obtained by modifying the heavy chain of a neurotoxin produced by Clostridium botulinum, wherein,

(i) the protein binds to nerve cells with higher or lower affinity than the native neurotoxin;

(ii) said protein having enhanced or reduced neurotoxicity as compared to the native neurotoxin, said neurotoxicity being suitably determined by a semiseptal assay; and/or the like and/or,

(iii) the protein exhibits lower affinity for neutralizing antibodies than native neurotoxin.

2. The transporter protein according to claim 1, wherein the neutralizing antibody inhibits the binding of native neurotoxin to a protein receptor or to a ganglioside receptor and/or inhibits the uptake of neurotoxin in nerve cells.

3. The transporter protein according to claim 1 or 2, wherein the transporter protein is taken up by a cell by endocytosis.

4. Transporter protein according to any one of claims 1 to 3, wherein said protein specifically binds to a molecule associated with the cytoplasmic membrane, a transmembrane protein, synaptobrevin, a protein of the synaptotagmin family, or synaptobotagmin 2(SV2), and/or synaptotagmin I and/or synaptotagmin II (cholinergic motor neurons) and/or SV2A, SV2B or SV2C, preferably human synaptotagmin I and/or human synaptotagmin II and/or human SV2A, SV2B or SV 2C.

5. Transporter protein according to any of claims 1 to 4, wherein the protein exhibits an affinity which is at least 15% higher or at least 15% lower than the native neurotoxin, preferably at least 50%, particularly preferably at least 80%, and particularly preferably at least 90%.

6. Transport protein according to any of claims 1 to 5, characterized in that the H of the transport protein_C-the fragment comprises at least one substitution and/or deletion, and/or insertion, and/or addition, and/or post-translational modification of a natural or unnatural amino acid, such that the affinity is increased or decreased compared to the natural neurotoxin.

7. The transporter protein according to any one of claims 1 to 6, wherein the neurotoxin is botulinum neurotoxin serotype A to G.

8. Transporter according to claim 7, characterized in that at least one amino acid at an amino acid position in the following proteins is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or non-natural amino acid:

positions 867 to 1296 of botulinum neurotoxin serotype A,

positions 866 to 1291 of botulinum neurotoxin serotype B,

864 to 1291, or 1280, of botulinum neurotoxin serotype C1,

positions 860 to 1276, or 1285 of botulinum neurotoxin serotype D,

the botulinum or Clostridium butyricum neurotoxin serotype E at positions 843 to 1251, or 1252,

botulinum or Clostridium barathetii neurotoxin serotype F positions 861 to 1274, 862 to 1280 or 1278 and 854 to 1268,

botulinum neurotoxin serotype G861-1297.

9. Transporter protein according to any preceding claim, wherein the native neurotoxin is neurotoxin serotype a, the transporter protein preferably binding to synaptobrevin 2(SV2), particularly preferably binding to SV2A, SV2B or SV 2C.

10. The transporter protein according to claim 9, wherein at least one amino acid at the following amino acid positions in the botulinum neurotoxin serotype a protein sequence is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted by a natural or non-natural amino acid: threonine at position 1195, arginine at position 1196, glutamic acid at position 1199, lysine at position 1204, isoleucine at position 1205, leucine at position 1206, serine at position 1207, leucine at position 1209, aspartic acid at position 1213, leucine at position 1217, phenylalanine at position 1255, arginine at position 1256, isoleucine at position 1258 and/or lysine at position 1260.

11. Transporter protein according to claim 10, characterized in that at least one amino acid at the following amino acid positions is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted by a natural or unnatural amino acid: arginine at position 1196, glutamic acid at position 1199, serine at position 1207, phenylalanine at position 1255, isoleucine at position 1258, and/or lysine at position 1260.

12. The transporter protein according to any of claims 10 or 11, wherein serine at position 1207 is substituted with arginine or tyrosine.

13. The transporter protein according to any one of claims 10 or 11, wherein the lysine 1260 is substituted with glutamic acid.

14. The transporter protein according to any one of claims 1 to 8, wherein the neurotoxin is botulinum neurotoxin serotype B, and wherein the transporter protein preferably binds to synaptotagmin I or II.

15. The transporter protein according to claim 14, wherein at least one amino acid at the following amino acid positions in the botulinum neurotoxin serotype B protein sequence is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted by a natural or non-natural amino acid: lysine at position 1113, aspartic acid at position 1114, serine at position 1116, proline at position 1117, valine at position 1118, threonine at position 1182, tyrosine at position 1183, phenylalanine at position 1186, lysine at position 1188, glutamic acid at position 1191, lysine at position 1192, leucine at position 1193, phenylalanine at position 1194, phenylalanine at position 1204, phenylalanine at position 1243, glutamic acid at position 1245, lysine at position 1254, aspartic acid at position 1255 and tryptophan at position 1256.

16. The transporter protein according to claim 15, wherein at least one amino acid at the following amino acid positions in the botulinum neurotoxin serotype B protein sequence is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or non-natural amino acid: valine at position 1118, tyrosine at position 1183, glutamic acid at position 1191, lysine at position 1192, glutamic acid at position 1245 and tryptophan at position 1256.

17. The transporter protein according to claim 16, wherein the tyrosine at position 1183 is substituted with leucine.

18. The transporter protein according to claim 16, wherein the glutamic acid at position 1191 is substituted with leucine.

19. The transporter protein according to any one of claims 1 to 8, wherein the neurotoxin is botulinum neurotoxin serotype G.

20. The transporter protein according to claim 19, wherein at least one amino acid at the following amino acid positions in the botulinum neurotoxin serotype G protein sequence is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or non-natural amino acid: phenylalanine at position 1121, lysine at position 1123, alanine at position 1124, serine at position 1125, methionine at position 1126, valine at position 1190, leucine at position 1191, serine at position 1194, glutamic acid at position 1196, threonine at position 1199, glutamic acid at position 1200, leucine at position 1201, phenylalanine at position 1202, phenylalanine at position 1212, phenylalanine at position 1248, lysine at position 1250, aspartic acid at position 1251, and tyrosine at position 1262.

21. The transporter protein according to claim 20, wherein at least one amino acid at the following amino acid positions in the botulinum neurotoxin serotype G protein sequence is post-translationally modified, and/or added, and/or deleted, and/or inserted, and/or substituted with a natural or non-natural amino acid: methionine at position 1126, leucine at position 1191, threonine at position 1199, glutamic acid at position 1200, lysine at position 1250, and tyrosine at position 1262.

22. The transporter protein of claim 21, wherein the tyrosine at position 1262 is substituted with a phenylalanine.

23. A composition comprising a transporter protein according to any one of claims 1 to 22 and at least one intervening molecule.

24. The composition of claim 23, wherein the intervening molecule is covalently bound to the transporter protein by a peptide bond, an ester bond, an ether bond, a sulfide bond, a disulfide bond, or a carbon-carbon bond.

25. The composition of claim 23 or 24, wherein the intervening molecule is a small organic molecule, a peptide, or a protein.

26. The composition of claim 25, wherein the small organic molecule is a viral inhibitor, a cytostatic agent, an antibiotic, or an immunoglobulin.

27. The composition of claim 25, wherein the protein is a protease.

28. The composition of claim 27, wherein the protease comprises Light Chains (LCs) of one or more botulinum neurotoxin serotypes A, B, C1, D, E, F and G.

29. The composition according to claim 27, wherein the protease comprises a proteolytically active fragment derived from the Light Chain (LC) of botulinum neurotoxin serotype A, B, C1, D, E, F or G, characterized by a proteolytic activity of at least 0.01%, preferably at least 50%, of the native protease.

30. The composition of claim 28 or 29, wherein the protease specifically cleaves a specific substrate within a cholinergic motor neuron.

31. The composition of claim 30, wherein the substrate is selected from the group consisting of a protein involved in neurotransmitter release in a neural cell and a protein capable of catalyzing a reaction within a neural cell.

32. The composition of claim 28 or 29, wherein the protease and the transporter are covalently bound by an amino acid sequence that is specifically recognized and cleaved by an endopeptidase.

33. The composition of claim 32 wherein the amino acid sequence comprises the sequence CXXXZKTKSLVPRGSKBXXC, wherein X is any desired amino acid and Z and B are each independently selected from the group consisting of alanine, valine, serine, threonine, and glycine.

34. The composition of claim 32, wherein upon specific cleavage by an endopeptidase, a disulfide bridge links the protease and the transporter prior to formation of the active holotoxin.

35. A pharmaceutical composition comprising a transporter protein according to any one of claims 1 to 22 or a composition according to any one of claims 23 to 34, optionally further comprising a pharmacologically acceptable excipient, diluent and/or additive.

36. Use of the pharmaceutical composition according to claim 35 for the treatment of dysfunctions and diseases that would otherwise be treated with botulinum neurotoxin.

37. The use according to claim 36, wherein the dysfunction or disease is one of: hemifacial spasm, spasmodic torticollis, blepharospasm, spasticity, dystonia, migraine, pain, cervical and lumbar spine dysfunction, strabismus, salivation, wound healing, snoring and depressive disorders.

38. A cosmetic composition comprising a transporter protein according to any one of claims 1 to 22 or a composition according to any one of claims 23 to 34, optionally together with cosmetic excipients, diluents and/or additives.

39. Use of the cosmetic composition according to claim 38 for treating hyperhidrosis and facial wrinkles for cosmetic purposes.

40. A process for the preparation of a transporter protein according to any one of claims 1 to 22 or a composition according to any one of claims 23 to 34 by recombination according to known methods.

41. The process for the preparation of a transporter protein according to claim 40, wherein H is_CThe gene of the fragment is flanked by two restriction endonuclease interfaces containing nucleic acid, which are linked to the other H of the botulinum neurotoxin_CThe endonuclease interfaces of the fragments are consistent, making them amenable to modular replacement while maintaining amino acid sequence similarity.

42. The process for preparing a composition according to claim 40, wherein the protease is genetically flanked by two nucleic acid-containing restriction endonuclease interfaces to other H's of the botulinum neurotoxin_CThe endonuclease interfaces of the fragments are consistent, making them amenable to modular replacement while maintaining amino acid sequence similarity.

43. A host cell comprising a recombinant expression vector, wherein the expression vector encodes a transporter protein according to any one of claims 1 to 22, or encodes a composition according to any one of claims 23 to 34.

44. The host cell according to claim 43, wherein the host cell is an E.coli cell, in particular E.coli K12, Saccharomyces cerevisiae, Pichia pastoris or Bacillus subtilis (Bacillus megaterium).

45. An expression vector comprising a nucleic acid encoding a transporter protein according to any one of claims 1 to 22 or a composition according to any one of claims 23 to 34.