Topical Compositions and Uses

Abstract

Chimeric polypeptides are provided that include one or more zinc finger motifs fused to a therapeutic peptide, such as a botulinum toxin (BoNT). [Solution] Provided is a chimeric polypeptide comprising a first zinc finger motif located at the C-terminus of a BoNT light chain or at the C-terminus of a BoNT heavy chain, and the BoNT heavy chain is located at the C-terminus of the BoNT light chain or is linked to the BoNT light chain via a disulfide bond. The chimeric polypeptide can comprise two or more zinc finger motifs and can be efficiently delivered transdermally to a subject.

Description

[Background technology]

[0001] Botulinum neurotoxins (BoNTs) are naturally occurring neurotoxins produced by Clostridium botulinum and related species. There are seven known serotypes of BoNTs: A, B, C1, D, E, F, and G. BoNTs are released from the bacteria as single polypeptide chains that subsequently autocleave into a 100 kDa heavy chain and a 50 kDa light chain linked by a single disulfide bond. The heavy chain targets the botulinum toxin to presynaptic nerve terminals and mediates internalization of the light chain into the cytoplasm. The botulinum light chain is a metalloprotease that specifically cleaves SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) complex proteins.

[0002] SNAREs are a large protein superfamily with over 60 members in mammalian cells. The core SNARE complex is a four-α-helical bundle that contains one helix each from synaptobrevin (also called vesicle-associated membrane protein, VAMP) and syntaxin-1, and two helices from synaptosomal associated protein 25 (SNAP-25). The four helices from these SNARE proteins wrap around each other to form a coiled-coil quaternary structure. Syntaxin-1 binds to the N-terminal helix of SNAP-25, and the synaptobrevin helix binds to the C-terminal helix of SNAP-25. The primary role of the SNARE complex is to mediate vesicle fusion, e.g., the fusion of synaptic vesicles with the presynaptic membrane in neurons.

[0003] BoNT specifically cleaves one of three core SNARE proteins: synaptobrevin, syntaxin-1, or SNAP-25. Inactivation of any of these three proteins disrupts the formation of the core SNARE complex or its interaction with other components in the SNARE supercomplex. Blocking the function of the SNARE supercomplex prevents the fusion of vesicles with the plasma membrane, thereby inhibiting the release of the neurotransmitter acetylcholine from the axon terminal, leading to muscle paralysis.

[0004] BoNT is the most potent natural toxin on earth, and as little as 50 ng of material is needed to cause human botulism. In nature, BoNT mainly infects wild and domestic animals and spreads through invertebrates. Depending on the origin of the toxin, human botulism is divided into five classes: food-borne botulism, infant botulism, inhalation botulism, iatrogenic botulism (resulting from excessive clinical doses of BoNT), and wound botulism (resulting mainly from drug injections). The first two are the most commonly seen human botulism.

[0005] The therapeutic use of BoNT was first proposed by Alan B. Scott in the late 1960s and was implemented in 1977 for children with strabismus. The extraordinary specificity of BoNT makes it an effective drug for human diseases characterized by hyperactivity of nerve endings. The largest proportion of therapeutic uses of BoNT are for neurological disorders such as dystonia, spasticity, hemifacial spasm, hyperhidrosis (excessive sweating), and hypersalivation (excessive saliva). Another important application of BoNT is the treatment of urological disorders such as detrusor-sphincter dystonia, idiopathic detrusor overactivity, neurogenic detrusor overactivity, urinary retention, anal fissure, and benign prostatic hyperplasia. Although rare, BoNT can be used to treat gastrointestinal and otorhinolaryngological disorders. BoNTA was approved by the United States (US) Food and Drug Administration (FDA) in 2002 for the treatment of moderate to severe glabellar furrows in adult patients aged 65 years or older. Since 2005, BoNTA has become the most widely used non-invasive physician-assisted cosmetic procedure. With an overall patient satisfaction rate of over 80%, injectable BoNTA is associated with the following problems: pain, injection marks, tenderness, bleeding, and There are many drawbacks, including bruising. In particular, injections of BoNTA into the crow's feet or lateral canthus area can carry a high risk of bruising due to the thin skin and superficial blood vessels. Some treatments require multiple injections to achieve maximum effectiveness. Summary of the Invention

[0006] In one embodiment, the invention provides a chimeric polypeptide comprising (a) a botulinum toxin (BoNT) light chain and (b) a first zinc finger motif located at the C-terminus of the BoNT light chain or at the C-terminus of the BoNT heavy chain, wherein the BoNT heavy chain is located at the C-terminus of the BoNT light chain or is linked to the BoNT light chain via a disulfide bond. In some embodiments, the chimeric polypeptide comprises 2000 or fewer amino acid residues.

[0007] In some embodiments, the BoNT light chain and the first zinc finger motif are on the same peptide chain. In some embodiments, the BoNT light chain and the first zinc finger motif are on different peptide chains.

[0008] In some embodiments, the chimeric polypeptide further comprises a second zinc finger motif located at the N-terminus of the BoNT light chain. In some embodiments, at least one of the first zinc finger motif and the second zinc finger motif is linked to at least one other finger motif. In some embodiments, at least one of the zinc finger motifs is a Cys2-His2 zinc finger motif. In some embodiments, the zinc finger motif comprises at least one alanine at residue 1, 2, 3, or 6 of the alpha-helical fragment of the zinc finger motif.

[0009] In one exemplary embodiment, a two-chain polypeptide is provided that includes (a) a first chain that includes a botulinum toxin (BoNT) light chain and three zinc finger motifs located at the N-terminus of the BoNT light chain, and (b) a second chain that includes a BoNT heavy chain and three zinc finger motifs located at the C-terminus of the BoNT heavy chain, the BoNT light chain being linked to the BoNT light chain via a disulfide bond. Similarly, in one embodiment, a chimeric polypeptide is provided that includes, from N-terminus to C-terminus, the three zinc finger motifs, the botulinum toxin (BoNT) light chain, the botulinum toxin (BoNT) heavy chain, and three additional zinc finger motifs.

[0010] In some embodiments, at least one of the zinc finger motifs comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 5-7. In some embodiments, the zinc finger motif comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first zinc finger is no more than 200 amino acid residues away from the C-terminus of the BoNT light chain or the BoNT heavy chain.

[0011] In some embodiments, the BoNT is selected from BoNT A, B, C, D, E, F, G, or a variant having at least 90% sequence identity thereto. In some embodiments, the BoNT is selected from BoNT subtypes A1-A10, B1-B8, E1-E9, and F1-F7. In some embodiments, the BoNT is BoNT A. In some embodiments, the BoNT light chain comprises an amino acid sequence of SEQ ID NO:8, or an amino sequence having at least 90% sequence identity to SEQ ID NO:8. In some embodiments, the BoNT heavy chain comprises an amino acid sequence of SEQ ID NO:9, or an amino sequence having at least 90% sequence identity to SEQ ID NO:9.

[0012] In some embodiments, the chimeric polypeptide is selected from the group consisting of SEQ ID NOs: 10-17. or a double-chain polypeptide processed from said amino acid chain.

[0013] Also provided in one embodiment is a chimeric polypeptide comprising a botulinum toxin (BoNT) light chain and at least one zinc finger motif. In some embodiments, the chimeric polypeptide comprises 2000 or fewer amino acid residues. In some embodiments, the zinc finger motif is located at the C-terminus of the BoNT light chain and is no more than 200 amino acid residues away from the BoNT light chain.

[0014] Another embodiment of the present invention provides a chimeric polypeptide comprising a therapeutic peptide and at least one zinc finger motif located at the C-terminus of the therapeutic peptide, wherein the N-terminus of the zinc finger motif is no more than 100 amino acid residues away from the C-terminus of the therapeutic peptide. In some embodiments, the therapeutic peptide is selected from the group consisting of epidermal growth factor (EGF) and superoxide dismutase (SOD).

[0015] Uses for the manufacture of medicaments and methods of treatment are also provided. In some embodiments, methods of administering a BoNT light chain to a mammalian subject are provided, comprising topically applying a formulation comprising a chimeric BoNT polypeptide of the invention. In some embodiments, administration can alternatively be intramuscular.

[0016] In some embodiments, the topical application is to the skin or mucous membranes of the eye, ear, nose, mouth, lips, urethral meatus, anus, or tongue. In some embodiments, the topical application is to a disrupted stratum corneum of skin. In some embodiments, the disruption is made with a needle or microneedle used to deliver the formulation. In some embodiments, the topical application is to the skin of the face or neck of a subject. In some embodiments, the formulation comprises a cream, gel, or spray.

[0017] In some embodiments, the subject is in need of treatment for facial wrinkles, dystonia, spasticity, hemifacial spasm, hyperhidrosis, or hypersalivation.

[0018] Also provided, in certain embodiments, are polynucleotides encoding the chimeric polypeptides of the invention, cells comprising the polynucleotides and / or the polypeptides, and formulations comprising the polypeptides. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the structures of various BoNTA fusion proteins tested in the examples. [Diagram 2] Figure 2, panels A and B, show the purification results of the BoNTA-CPP recombinant fusion protein expressed from E. coli. (A) TAT and Pep-1 did not produce good protein expression and purification yields. (B) BoNTA-ZFP (protein number 6) produced good protein expression and purification yields. [Diagram 3]Figure 3 shows the results of a SNAPtide assay of the in vitro activity of cell-permeable BoNTA protein. The positive control is a commercially available recombinant BoNTA light chain (BoNTA-LC, R&D Systems). Mock is a reaction with SNAPtide only. [Figure 4] Figure 4 shows the results of a SNAPtide assay of cytolysis of human dermal fibroblasts treated with cell-permeable BoNTA protein. The negative control is commercially available recombinant BoNTA-LC (R&D Systems). [Diagram 5] FIG. 5 shows representative images of the in vivo effects of cell-permeable BoNTA protein. [Figure 6] Figure 6 is a scatter plot of digit abduction in mice treated with cell-permeable BoNTA protein. A: treated with 0.9% NaCl saline. B: injected with BOTOX. C: pre-treated with microneedle followed by treatment with BOTOX. D: pre-treated with microneedle followed by treatment with cell-permeable BoNTA-ZFP (protein #6). E: treated with cell-permeable BoNTA-ZFP (protein #6) without pre-treated with microneedle. [Figure 7] Figure 7 shows expression of BoNTA-ZFP (Protein No. 6) in insect cells. M: protein marker. T: total cell lysate. S: soluble fraction. +: loading control. [Figure 8] FIG. 8 shows the results of a treadmill experiment to analyze the muscle paralyzing effect of BoNTA-ZFP. [Figure 9] FIG. 9 shows the results of a balance beam experiment to analyze the muscle paralyzing effects of BoNTA-ZFPs. [Figure 10] FIG. 10 shows the results of a footprint experiment to analyze the muscle paralyzing effect of BoNTA-ZFP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0021] As used herein, the term "polypeptide" is intended to encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain(s) of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to a chain(s) of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides may be generated in any manner, including chemical synthesis.

[0022] As used herein, the term "isolated" with respect to a cell, a nucleic acid such as DNA or RNA, refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. Also, as used herein, the term "isolated" refers to a nucleic acid or peptide that is substantially free of cellular material, bacterial material, or culture medium when produced by recombinant DNA technology, or precursor chemicals or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. Also, the term "isolated" is used herein to refer to a cell or polypeptide that is isolated from other cellular proteins or tissues. Isolated polypeptides are intended to encompass both purified and recombinant polypeptides.

[0023] As used herein, the term "recombinant" refers to a polypeptide or polynucleotide. " refers to a species of polypeptide or polynucleotide that does not occur in nature; a non-limiting example is that polynucleotides or polypeptides that do not normally occur together can be made by combining them.

[0024] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matched or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity with one of the sequences of the invention, preferably less than 25% identity.

[0025] When a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a particular percentage (e.g., 60%, 65%, 70%, 75%, 8%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, it means that, when aligned, that percentage of bases (or amino acids) are the same in comparison of the two sequences.

[0026] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a degree of homology or sequence identity with the nucleotide sequence of a nucleic acid or its complementary strand. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that has a degree of homology with a double-stranded nucleic acid or its complementary strand. In one embodiment, a homolog of a nucleic acid can hybridize with a nucleic acid or its complementary strand. Similarly, an "equivalent polypeptide" refers to a polypeptide that has a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, an equivalent polypeptide or polynucleotide has 1, 2, 3, 4, or 5 additions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some embodiments, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0027] Hybridization reactions can be carried out under conditions of different "stringency". Generally, low stringency hybridization reactions are carried out at about 40° C. in a solution of about 10×SSC or equivalent ionic strength / temperature. Medium stringency hybridizations are typically carried out at about 50° C. in about 6×SSC, and high stringency hybridizations are generally carried out at about 60° C. in about 1×SSC. Alternatively, hybridization reactions can be carried out under "physiological conditions", as known to those of skill in the art. Non-limiting examples of physiological conditions include the temperature, ionic strength, pH, and Mg concentration normally found within a cell.2+ is the concentration.

[0028] A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) instead of thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term "polymorphism" refers to the coexistence of more than one form of a gene or part thereof. A part of a gene that has at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region of a gene." A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.

[0029] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute a double-stranded form.

[0030] The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if, either in nature or when manipulated by methods known to those of skill in the art, it is capable of being transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complementary strand of such a nucleic acid, from which the coding sequence can be deduced.

[0031] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a second thiol group.

[0032] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose being to prevent or slow (reduce) undesirable physiological changes or disorders, such as undesirable wrinkles. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, a stabilized (i.e., not worsening) state of the disease, delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if not treated. Those in need of treatment include those already with the condition or disorder, as well as those prone to having the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0033] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0034] As used herein, phrases such as "to a patient in need of treatment" or "subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of an antibody or composition of the invention used, for example, for detection, for diagnostic procedures, and / or for treatment.

[0035] Chimeric polypeptides having zinc finger motifs There is a need in the market for transdermal delivery of BoNT proteins. Transdermal delivery of proteins is inherently challenging, especially through the skin. Skin is composed of two layers of cells known as the epidermis and dermis. The epidermis, the top layer of skin, is a stratified squamous epithelium composed of basal and differentiated keratinocytes. Keratinocytes are the major cell type of the epidermis. Keratinocytes in the basal layer proliferate through mitosis and undergo multiple cell differentiation steps to become anucleated cells. Anucleated or differentiated keratinocytes are highly organized tissue structures that secrete keratin proteins and lipids that provide a protective barrier against invading agents such as pathogens.

[0036] Development of transdermal BoNT compositions has not been successful. However, the present invention shows that BoNT can be made to penetrate the skin by fusing BoNT polypeptides to one or more zinc finger motifs (or zinc finger protein domains, or ZFPs). Zinc finger proteins are natural transcription factors that can be reprogrammed to recognize target genomic loci. Zinc finger nuclease-chimeric proteins contain an N-terminal ZFP domain and a C-terminal Fok I endonuclease domain and have been shown to be inherently cell-permeable. Some ZFPs contain a Cys2-His2 domain. Cys2-His2ZFPs are composed of about 30 amino acids with a ββα structure.

[0037] This finding is surprising and unexpected for at least the following reasons: First, the ability to efficiently deliver large proteins across animal skin is itself surprising and unexpected. In particular, the cell-penetrating activity of ZFPs was known to be energy dependent. This meant that it was predicted to be only limited, if at all, efficient in the stratum corneum, the outermost layer of skin, which is formed from dead corneocytes, which, even when disrupted by physical or chemical means, still constitute an insurmountable barrier to typical biological molecules.

[0038] Second, ZFPs are zinc-dependent metalloproteases, and botulinum toxin activity is also known to require zinc, as it can be inhibited by protease inhibitors and zinc chelators. Because both ZFPs and BoNTs require the uptake of zinc ions, it was suspected that the presence of both ZFPs and BoNTs in the fusion protein would cause interference between them, thereby reducing or even inactivating their activity.

[0039] In this regard, the inventors envisioned that such interference could be avoided or reduced by fusing the ZFP toward the C-terminus of the BoNT. Additionally or alternatively, the use of bipartite (tandem) ZFPs could ensure that ZFP activity is maintained.

[0040] Third, as demonstrated in the Examples, BoNT-ZFP fusion proteins were expressed in large amounts from bacterial cells, much higher than when BoNT was fused to two commonly studied cell-penetrating peptides, TAT or Pep-1, an effect that was unsuggested and unexpected.

[0041] Finally, the expressed BoNT-ZFP fusion proteins not only exhibited high enzymatic activity (see, e.g., FIG. 3), but also retained said activity after crossing cellular boundaries and entering the intracellular space (see, e.g., FIG. 4). Furthermore, when used in an in vivo setting, BoNTA-ZFP fusion proteins applied topically, preferably to the skin of animals treated with microneedles, produced muscle paralysis similar to injected BoNTA. Such a therapeutic effect can be achieved with a topical formulation. This was also surprising as this was not anticipated in the art.

[0042] Thus, in one embodiment of the invention, there is provided a chimeric (or fusion) polypeptide comprising: (a) a botulinum toxin (BoNT) light chain; and (b) a zinc finger motif located at the C-terminus of the BoNT light chain.

[0043] Also provided in one embodiment is a chimeric (or fusion) polypeptide comprising: (a) a botulinum toxin (BoNT) light chain; (b) a BoNT heavy chain; and (c) a zinc finger motif located at the C-terminus of the BoNT heavy chain. In some embodiments, the light chain and the heavy chain are on the same peptide chain. In some embodiments, the light chain is N-terminal to the heavy chain. In some embodiments, the light chain is C-terminal to the heavy chain. In some embodiments, the light chain and the heavy chain are on different peptide chains and are linked by disulfide bonds.

[0044] Also provided in some embodiments are chimeric polypeptides comprising a botulinum toxin (BoNT) light chain and at least one zinc finger motif, hi one embodiment, the chimeric polypeptide further comprises a BoNT heavy chain.

[0045] In some embodiments, the overall size of the chimeric polypeptide is 5,000 amino acid residues or less, alternatively 4,000 amino acid residues or less, 3,000 amino acid residues or less, 2,000 amino acid residues or less, 1,800 amino acid residues or less, 1,600 amino acid residues or less, 1,500 amino acid residues or less, 1,400 amino acid residues or less, 1,300 amino acid residues or less, 1,200 amino acid residues or less, 1,100 amino acid residues or less, 1,000 amino acid residues or less, 900 amino acid residues or less, 800 amino acid residues or less, 700 amino acid residues or less, 600 amino acid residues or less, 500 amino acid residues or less, 450 amino acid residues or less, 400 amino acid residues or less, 350 amino acid residues or less, 300 amino acid residues or less, 250 amino acid residues or less, or 200 amino acid residues or less.

[0046] There are at least seven types of botulinum toxins, designated types AG. Types A and B can cause human disease and are used commercially and medically. Types C-G are less common. Botulinum toxin types A and B are used in medicine to treat various muscle spasms and diseases characterized by overactive muscles. Each BoNT serotype may have subtypes. For example, the following subtypes are known: BoNT A1-A10, B1-B8, E1-E9, F1-F-7.

[0047] The term BoNT, or a particular type of BoNT, or a subtype of BoNT, also encompasses their equivalent polynucleotides, e.g., polynucleotides having a certain level of sequence identity (e.g., at least 85%, 90%, 95%, 98%, or 99%), or polynucleotides modified by the addition, deletion, or substitution of one or more amino acid residues. In some embodiments, the substitution is a conservative amino acid substitution.

[0048] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, tyrosine, cysteine), and amino acids with β-branched side chains (e.g., threonine, tyrosine, cysteine). Examples of amino acid residues that may be used include amino acids with essential side chains (e.g., leonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in a polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a chain of amino acids can be replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0049] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or greater indicates a conservative substitution between the two amino acids. [Table 1-1] [Table 1-2]

[0050] In some embodiments, the BoNT peptide includes only one, only two, or only three of the above substitutions from a native BoNT peptide.

[0051] Non-limiting examples of BoNT light chains include SEQ ID NO:8 (BoNT A light chain) and amino sequences having at least 90% (alternatively, at least 95%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:8. Non-limiting examples of BoNT heavy chains include SEQ ID NO:9 (BoNT A heavy chain) and amino sequences having at least 90% (alternatively, at least 95%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:9. The amino acid sequences of SEQ ID NOs:8 and 9 are provided in Table 2 below. [Table 2]

[0052] A "zinc finger motif" is a small protein structural motif characterized by the coordination of one or more zinc ions to stabilize the fold. Generally, zinc fingers coordinate a zinc ion to a combination of cysteine ​​and histidine residues. The number and order of these residues can be used to classify different types of zinc fingers (e.g., Cys2His2, Cys4, and Cys6). Yet another method classifies zinc finger proteins into fold groups based on the overall shape of the protein backbone within the folded domain. The most common fold groups of zinc fingers include Cys2His2 (classical zinc finger), treble clef, zinc ribbon, gag knuckle, Zn2 / Cys6, and TAZ2 domain.

[0053] The Cys2His2 fold group adopts a simple ββα fold and contains the amino acid sequence motif: X2-Cys-X 2,4 -Cys-X 12 -His-X 3,4,5 -His.

[0054] Individual zinc finger domains can occur as tandem repeats, with two, three, or more fingers comprising the DNA binding domain of the protein.

[0055] Zinc finger motifs can be modified to eliminate or reduce DNA binding ability. For example, modified Cys2His2 contains at least an alanine at residue 1, 2, 3, or 6 of the alpha-helical fragment of the zinc finger motif. Non-limiting examples of zinc finger motifs are shown in Table 3 below. Some of the sequences in Table 3, SEQ ID NOs: 1 and 5-7, are single zinc finger motifs, while a few others (tandems of zinc finger motifs), SEQ ID NOs: 2-4, contain multiple linked zinc finger motifs. When two or more zinc fingers are used in tandem, they can be located immediately next to each other or linked via a peptide linker, i.e., short peptides ranging from 1, 2, or 3 amino acid residues to 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in length. The modified alanine residues in SEQ ID NO: 1 are underlined and shown in bold. [Table 3]

[0056] In some embodiments, the chimeric polypeptide further comprises a zinc finger motif located N-terminally of the BoNT light chain. In some embodiments, the chimeric polypeptide further comprises a zinc finger motif N-terminally of the BoNT heavy chain. In some embodiments, the chimeric polypeptide is a di-chain polypeptide and comprises a zinc finger motif (or a tandem of two zinc fingers or three zinc fingers) C-terminally of the BoNT light chain. In some embodiments, the chimeric polypeptide is a di-chain polypeptide and comprises a zinc finger (or a tandem of two zinc fingers or three zinc fingers) C-terminally of the BoNT light chain and a zinc finger (or a tandem of two zinc fingers or three zinc fingers) N-terminally of the BoNT heavy chain.

[0057] In some embodiments, at least one of the zinc finger motifs is linked to at least one or more zinc finger motifs to form a bipartite or tripartite zinc finger motif (e.g., a tandem zinc finger motif), which in some embodiments have zero, one, two, three, four, or five amino acid residues between them.

[0058] The distance between the BoNT light chain, heavy chain, and zinc finger motifs can be adjusted as desired or necessary. In some embodiments, the zinc finger is separated by 200 amino acid residues or less from the N-terminus or C-terminus of the adjacent BoNT light or heavy chain. In some embodiments, the distance is 0 to about 150, 5 to 100, 10 to 75, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 150, 20 to 100, 20 to 50, or 50 to 100 amino acid residues. In some embodiments, the distance is provided by inserting a spacer sequence (e.g., of alanine, glycine, or a combination thereof).

[0059] Non-limiting examples of chimeric polypeptide sequences are provided in SEQ ID NOs: 10-17 (Table 3). Some of these sequences contain both a BoNT light chain and a BoNT heavy chain, and these sequences encompass both the uncleaved single-chain and the uncleaved two-chain forms. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0060] Furthermore, it is believed that the present technology can be applied to other therapeutic peptides, such as epidermal growth factor (EGF) and superoxide dismutase (SOD), without limitation. Thus, in one embodiment, the present invention provides a chimeric polypeptide comprising a therapeutic peptide and at least one zinc finger motif. In some embodiments, the zinc finger motif is located C-terminal to the therapeutic peptide. In some embodiments, the zinc finger motif is separated from the therapeutic peptide by no more than 100 amino acid residues (or no more than 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 amino acid residues). In some embodiments, the zinc finger motif is linked to another zinc finger motif to form a bi-element zinc finger motif. In some embodiments, the chimeric polypeptide comprises zinc finger motifs (or bi-element zinc finger motifs) at both the N-terminus and C-terminus of the therapeutic peptide.

[0061] In some embodiments, the therapeutic peptide is selected from EGF and SOD, example sequences of which are shown in Table 5. Also described herein are example zinc finger motifs, example spacer sequences, and distances between the zinc finger motif and the therapeutic peptide. [Table 5]

[0062] In some embodiments, the polypeptides may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, or PEG. The polypeptides may be conjugated or fused to therapeutic agents, which may include a detectable label, such as a radioactive label, immunomodulatory agents, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents, which may be drugs or toxins, ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0063] The polypeptide can be detectably labeled by coupling to a chemiluminescent compound. The presence or absence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence or absence of luminescence produced during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0064] The polypeptide also includes lanthanide series 152 They may also be detectably labeled with fluorescent metals, such as Eu or other metals, which can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0065] The invention also provides isolated polynucleotides or nucleic acid molecules encoding the polypeptides, variants or derivatives of the polypeptides of the invention. The polynucleotides of the invention can be present in any combination with the polypeptides, variants or derivatives thereof, on the same or separate polynucleotide molecule. Further, a polynucleotide of the invention may encode portions of the heavy and light chains of a polypeptide, variant or derivative thereof, either on the same or separate polynucleotide molecules.

[0066] The polynucleotide encoding the fusion polypeptide or a domain thereof can be inserted into an "expression vector". The term "expression vector" refers to a genetic construct, such as a plasmid, virus, or other vehicle known in the art, that can be designed to contain a polynucleotide encoding a polypeptide of the invention. Such an expression vector is typically a plasmid that contains a promoter sequence that promotes the transcription of the inserted genetic sequence in a host cell. An expression vector typically contains an origin of replication and a promoter, as well as a gene that allows phenotypic selection of transformed cells (e.g., an antibiotic resistance gene). A variety of promoters, including inducible and constitutive promoters, can be used in the present invention. Typically, an expression vector contains a replicon site and control sequences derived from a species compatible with the host cell.

[0067] Transformation or transfection of host cells with polynucleotides can be performed using conventional techniques well known to those skilled in the art. For example, if the host cell is E. coli, competent cells capable of incorporating DNA can be prepared using CaCl2, MgCl2, or RbCl methods known in the art. Alternatively, physical means such as electroporation or microinjection can be used. Electroporation can transfer polynucleotides into cells by high-voltage electrical impulses. Additionally, polynucleotides can be introduced into host cells by protoplast fusion using methods well known in the art. Suitable methods for transforming eukaryotic cells, such as electroporation and lipofection, are also known.

[0068] A "host cell" encompassed by the present invention is any cell in which a polynucleotide of the present invention can be used to express a fusion polypeptide or a functional domain thereof. The term also includes the progeny of the host cell. Useful host cells include bacterial cells (e.g., Clostridium botulinum), fungal cells (e.g., yeast cells), insect cells (e.g., Spodoptera), plant cells, and animal cells. The fusion polypeptides of the present invention can be produced by expressing a polynucleotide encoding the fusion polypeptide in prokaryotes. These include, but are not limited to, microorganisms such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA vector encoding the fusion polypeptide of the present invention. Introduction of the construct into the host cell can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation.

[0069] The constructs can be expressed in Clostridium botulinum, where BoNT proteins are naturally produced. It is a surprising discovery of the present invention that chimeric proteins containing the light and / or heavy chains of BoNT can be efficiently produced in insect cells (e.g., Spodoptera frugiperda Sf9). Thus, in one embodiment, the host cell can be an insect cell, such as a Lepidoptera cell, a Noctuidae cell, a Spodoptera cell, and a Mammalian armyworm cell.

[0070] For long-term, high-yield production of recombinant proteins, stable expression is usually used. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with a cDNA encoding the fusion polypeptide of the invention under the control of appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Selectable markers include those that inhibit selective killing. It confers resistance to the agent and allows the growth of resistant cells through stable integration of the heterologous polynucleotide. Such resistant cells proliferate to form foci that can then be cloned and expanded into cell lines.

[0071] Treatment Methods and Uses As described herein, the zinc finger motif-containing chimeric (fusion) polypeptides of the invention can be effectively delivered transdermally and thus have therapeutic uses, depending on the therapeutic protein fused to them.

[0072] Chimeric polypeptides comprising a BoNT light chain, optionally further comprising a BoNT heavy chain, have a wide range of cosmetic and therapeutic applications. In cosmetic applications, such chimeric polypeptides are useful for treating wrinkles and adjusting the corners of the mouth or the line of the upper lip. In therapy, chimeric polypeptides are useful for treating neurological disorders such as dystonia, spasticity, hemifacial spasm, hyperhidrosis (excessive sweating), and hypersalivation (excessive saliva). Chimeric polypeptides may also be used for urological disorders such as detrusor-sphincter dystonia, idiopathic detrusor overactivity, neurogenic detrusor overactivity, urinary retention, anal fissure, and benign prostatic hyperplasia. Further indications include gastrointestinal disorders, otorhinolaryngological disorders, or other medical conditions. In some embodiments, chimeric polypeptides are used for treating facial wrinkles, dystonia, spasticity, hemifacial spasm, hyperhidrosis, or hypersalivation.

[0073] In another example, chimeric polypeptides containing EGF can be used to treat conditions such as diabetic foot ulcers or in general wound healing. In yet another example, chimeric polypeptides containing SOD peptides can be used to reduce free radical damage to the skin, for example, fibrosis after radiation for breast cancer.

[0074] Topical application of the chimeric polypeptide can be to a dermatological site of a subject, such as the skin or mucous membranes of the eyes, ears, nose, mouth, lips, urethral meatus, anus, or tongue. Examples of specific areas include, but are not limited to, the face, neck, head, legs, shoulders, back, palms, feet, groin, armpits, elbows, arms, knees, buttocks, trunk, and pelvis. Similarly, the chimeric polypeptide can be administered intramuscularly in these areas.

[0075] Thus, in some embodiments, a method is provided for administering a therapeutic peptide (e.g., BoNT, EGF, or SOD) to a mammalian subject, comprising topically or intramuscularly applying a formulation comprising a chimeric peptide comprising a zinc finger motif and the therapeutic peptide to the mammalian subject.

[0076] In some embodiments, when topical application is to the stratum corneum of the skin, it is preferred that the stratum corneum is disrupted. Disruption of the stratum corneum may be accomplished using a needle, such as a microneedle, as shown in the examples. Alternatively, application can be intramuscular.

[0077] The specific dosage and treatment regimen for a particular patient will depend on a variety of factors, including the specific polypeptide, variant or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, excretion rate, drug combination, and the severity of the particular disease being treated. The judgment of such factors by a medical practitioner is within the ordinary skill of the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0078] composition The present invention also provides pharmaceutical compositions. Such compositions contain an effective amount of a chimeric polypeptide. The peptide comprises a peptide and an acceptable carrier.

[0079] In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. Federal or state government for use in animals, especially humans, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias. Furthermore, a "pharmaceutically acceptable carrier" generally refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or any auxiliary formulation.

[0080] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Also contemplated are antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and tonicity adjusters such as sodium chloride or dextrose. These compositions can take the form of gels, cremes, sprays, solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories, with traditional binders and carriers such as triglycerides.

[0081] Also provided in certain embodiments are kits and packages that include the chimeric polypeptide, or a composition or formulation thereof, and instructions for using the chimeric polypeptide, composition, or formulation. In some embodiments, the kit or package further includes a needle or microneedle for delivering the chimeric polypeptide, composition, or formulation. As discussed above, microneedles can help disrupt the stratum corneum of the skin, thereby improving delivery.

[0082] Animal Testing Methods Currently, rat digit abduction score (DAS) assay is the main physiological model for the evaluation of BoNT-induced skeletal muscle paralysis. This assay is not completely objective and prone to error. In some embodiments, the present invention describes a new method for quantitatively measuring BoNT-induced skeletal muscle paralysis in animals.

[0083] In one example, animals are placed on a treadmill before or after receiving a test agent. When the treadmill is started, the animals are forced to move as far as they are physically able with a maximum running time of 30 minutes. The duration, distance, or time of movement can quantitatively reflect the effect of the test agent (with appropriate controls, such as animals not receiving the test agent).

[0084] In another example, an animal is placed on a balance beam and guided to walk to the other end: muscle paralysis resulting in a reduced ability to maintain balance is reflected in the time it takes the animal to cross the beam.

[0085] In yet another example, the animal's footprints are measured and taken while standing or walking (such as on a treadmill). The size of the footprints can also be a quantitative measure of the degree of paralysis. EXAMPLES

[0086] Example 1: Expression and purification of BoNTA-CPP fusion protein from E. coli This experiment demonstrates that a BoNTA-CPP (cell penetrating peptide) fusion protein can be expressed and purified from E. coli cells.

[0087] method Protein expression The pET28b vectors containing the different BoNTA constructs (protein numbers 1-8, FIG. 1) were transformed into BL21(DE3) E. coli cells. The sequences of these constructs are shown in SEQ ID NOs: 10-17. His6: histidine tag; BoNTA-LC: BoNT A light chain; BoNTA-HC: BoNT A heavy chain; ZFP2: tandem zinc finger peptide (SEQ ID NO: 1); TAT: transcriptional activator of transcription peptide; Pep-1: pep-1 peptide. TAT and Pep-1 are known as cell-penetrating peptides (CPPs).

[0088] A single colony was picked from the agar plate and grown overnight at 37°C in 10 mL of lysogeny medium (LB medium) supplemented with 50 μg / mL kanamycin and 90 μM ZnCl2. The next day, the 10 mL starter culture was inoculated into 1 liter of LB medium supplemented with 50 μg / mL kanamycin and 90 μM ZnCl2 and grown to an OD 600 to 0.8. Protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 4 h at 25° C. Cell pellets were collected by centrifugation at 5,000 rpm for 10 min.

[0089] Protein purification Cell pellets (approximately 30 grams) from 2 liter cultures were resuspended in 200 mL of BoNTA lysis buffer (20 mM HEPES, pH 7.0, 500 μM NaCl, 0.01% Triton X-100, 1× protease mix (Roche), and 10% glycerol) and sonicated three times on ice. Lysed cells were centrifuged at 25,000 g for 1 h at 4°C, and the supernatant was transferred to a new tube. To this supernatant, 1 mL (settled volume) of equilibrated Ni-NTA resin (Qiagen) was added. BoNTA protein containing His6 tag was bound to the resin by rotating for 30 min. The resin was transferred to a column and the flow-through was discarded. The resin was washed with 50 mL of BoNTA wash buffer (20 mM HEPES, pH 7.0, 500 mM NaCl and 10% glycerol, 20 mM imidazole) and then eluted with 50 mL of BoNTA elution buffer (20 mM HEPES, pH 7.0, 500 mM NaCl and 10% glycerol, 300 mM imidazole). The eluted fraction was then concentrated and then diluted with start buffer (20 mM TrisHCl, pH 8.5) and end buffer (20 mM TrisHCl, pH 8.5, 1M The fractions with optimal purity were combined again, concentrated by spin concentration, and stored at -80°C after addition of 10% glycerol for further use.

[0090] Results and Data Analysis One-step affinity purification yielded protein with only modest purity. After a second ion exchange step, the purity was significantly improved. The total yield was estimated at 0.1 mg per liter of culture, and the purity of the final product was 90% (Figure 2, panels AB). Surprisingly, fusion proteins with ZFPs resulted in higher protein expression and purification than fusions with cell-penetrating peptides (TAT and Pep-1, Figure 2, panel A).

[0091] This example demonstrates that the BoNTA-CPP fusion protein can be expressed as a recombinant protein from E. coli and purified by affinity purification followed by ion exchange. This shows that it is possible.

[0092] Example 2: Cleavage of botulinum matrix by BoNTA-CPP fusion protein This experiment demonstrates that the expressed BoNTA-CPP fusion protein has activity towards the BoNT substrate SNAP-25.

[0093] Botulinum Activity Testing Using the SNAPtide™ Assay SNAPtide (Millipore, Cat. No. 567333-200NMOL) was diluted to 5 μM in reaction buffer (20 mM, pH 7.4, 0.25 mM ZnCl2, 5 mM DTT, 0.05% Tween-20). Each recombinant BoNTA-ZFP fusion protein (200 nM; protein no. 1–8) was added to the reaction buffer containing SNAPtide. The reaction was incubated at 37°C for 40 min. Fluorescence was recorded at an excitation wavelength of 320 nm and an emission wavelength of 420 nm using a plate reader. SNAPtide was a short peptide derived from SNAP-25, an intracellular substrate of BoNTA. SNAPtide contained the cleavage site of BoNTA and both a fluorescent dye group and a quenching group. Cleavage of the peptide liberated the fluorescent dye and activated the fluorescence. The reaction positive control was a commercially available recombinant BoNTA light chain (BoNTA-LC) protein (Cat. No. 4489-ZN-010) purchased from R&D Systems. All data were replicated in triplicate.

[0094] Results and Data Analysis Data in Figure 3 are presented as mean ± standard deviation and analyzed by one-tailed Student's t-test. All test samples, including commercial BoNTA-LC, had significantly (p<0.05) higher signals than the SNAPtide control group. As shown in Figure 3, both single-terminated and two-part fusions of ZFPs retained the activity of BoNTA against SNAP-25-derived peptide substrates.

[0095] Example 3: Cell-penetrating activity assay of BoNTA-ZFP fusion proteins This experiment demonstrates that selected BoNTA-ZFP fusion proteins can effectively penetrate human dermal fibroblasts (hDFs).

[0096] Transduction of hDF cells with recombinant BoNTA-ZFP fusion proteins hDF cells were seeded on polylysine-coated 6-well plates. 24 hours after seeding, cells were washed three times with phosphate-buffered saline (PBS). BoNTA-ZFP proteins (protein numbers 5 and 6) and control protein (R&D BoNTA-LC, commercial BoNTA light chain from R&D Systems) were diluted in DMEM serum-free medium. Cells were treated with BoNTA-ZFP proteins (0.15 μM) and BoNTA-LC (0.5 μM) for 2 hours at 37°C. Cells were then washed three times with PBS supplemented with 0.5 mg / mL heparin to remove surface-bound proteins and then harvested by trypsinization. Collected cells were lysed by sonication and BoNTA activity was analyzed as described above.

[0097] Results and Data Analysis The data shown in Figure 4 are presented as mean ± standard deviation and analyzed by one-tailed Student's t-test. hDF cells treated with BoNTA-ZFP proteins (proteins no. 5 and 6) showed obvious BoNTA activity with significantly higher signals (p<0.05) than the control group, indicating that BoNTA-ZFP proteins can effectively penetrate cells.

[0098] Example 4: In vivo activity of BoNTA-ZFP proteins in mice This experiment demonstrates that when applied to intact or microneedle-treated mouse skin, a BoNTA-ZFP fusion protein induces muscle paralysis characterized by digit abduction.

[0099] Finger abduction experiment Fifteen C57 female mice weighing approximately 36 g were randomly divided into five groups (n = 3). In all mice, the left leg was untreated as a control, and the right leg was treated with different drugs. Mice were anesthetized before treatment. In the mock group (A), mice were administered storage buffer (20 mM HEPES, pH 7.0, 300 mM NaCl and 10% glycerol). In the BOTOX (Allergan) injection group (B), BOTOX was reconstituted in 0.9% NaCl saline as indicated, and 5 μL of 45 U / mL solution was injected into the right leg. In group C, the legs and feet of mice were pretreated with a microneedle roller (RoHS MR20, 0.2 mm, house use), followed by topical application of 60 μL of 45 U / mL BOTOX. In group D, the legs and feet of mice were pretreated with a microneedle roller (RoHS MR20, 0.2 mm, house use) followed by topical application of 60 μL of 0.05 mg / mL BoNTA-ZFP protein (protein number 6) in storage buffer (20 mM HEPES, pH 7.0, 300 mM NaCl, and 10% glycerol). In group E, 60 μL of 0.05 mg / mL BoNTA-ZFP protein in storage buffer (20 mM HEPES, pH 7.0, 300 mM NaCl, and 10% glycerol) was topically applied. The microneedle roller treatment was applied by rolling the legs and feet three times. For topical application, the drug was evenly spread on the legs and feet, massaged, and then air-dried. This was repeated several times until all the solution was administered. Digital abduction was recorded after the mice woke up on day 0 and then daily for the following four days.

[0100] Results and Data Analysis Both injected BOTOX and BoNTA-ZFP with microneedle pretreatment showed notable digit abduction with scores significantly higher (p<0.05; Student's t-test) than control group A (Figure 5). Direct application of BoNTA-ZFP had minimal effect. Digital abduction scores (DAS) were assessed blinded by three separate investigators. DAS reached peak values ​​on day 2 (Figure 6). Thus, this example shows that when applied to mouse skin, cell-permeable BoNTA-ZFP causes muscle paralysis, indicating in vivo activity of NAP-25 cleavage.

[0101] Example 5: Expression in insect cells This experiment demonstrates that BoNTA-ZFP fusion proteins can be expressed in insect cells with high yields. Considering that natural BoNT is produced by the bacterium Clostridium botulinum (a prokaryote), the large-scale production of BoNTA from insect cells (a eukaryote) is quite surprising.

[0102] method The pFastBac vector containing protein number 6 (SEQ ID NO: 15) was packaged into baculovirus (BV). BV containing recombinant protein number 6 was used to infect Sf9 insect cells. Cells were harvested when cell viability was less than 60%. Approximately 1 mL of culture was centrifuged to collect cells. The cells were resuspended in 1 mL of PBS buffer and sonicated. Approximately 10 μL of cell lysate was resolved by SDS-PAGE. Proteins were detected with anti-His antibody and imaged.

[0103] Results and Data Analysis Soluble BoNTA-ZF represents nearly 100% of the total protein (Figure 7). The amount of protein was quantified at 1 μg compared to the loading control (5 μg protein). (Figure 7). The expression yield is therefore calculated to be 100 mg per liter of culture.

[0104] Example 6: Intramuscular injection produces muscle paralysis method Various doses of BoNTA-ZFP were injected intramuscularly into the right gastrocnemius muscle using a Hamilton syringe. Muscle paralysis was monitored as described in Example 4.

[0105] Results and Data Analysis Intramuscular injection of 1 or 3 ng of BoNTA-ZFP (Protein #6) resulted in clear digit abduction, with all mice reaching a score of 4 (n=3 per group).

[0106] Example 7: Treadmill testing In this example, the method for measuring the muscle paralysis effect of drugs is tested.Compared with the spontaneous ongoing study, this method can objectively reflect the muscle paralysis effect of cell-permeable BoNTA-ZFP.

[0107] method BoNTA-ZFP was topically applied to CJ57 female mice as described in Example 4. Mice were subjected to the following series of angle and speed settings: 0° / 5m / min, 3° / 8m / min, 6° / 11m / min, 9° / 14m / min, 12° / 17m / min, 12° / 20m / min. Mice were maintained at each step for 5 minutes. Total distance traveled was measured.

[0108] Results and Data Analysis Mice treated with Botox injections or topical BoNTA-ZFP showed impaired exercise performance in a treadmill experiment compared to buffered saline (FIG. 8).

[0109] Example 8: Balance beam test In this example, an alternative method for measuring the muscle paralyzing effects of drugs is examined.

[0110] method BoNTA-ZFP was applied topically to CJ57 female mice as described in Example 4. The mice were subjected to the balance beam experiment. The time it took the mice to cross the balance beam was measured.

[0111] Results and Data Analysis Mice treated with Botox injections or topical BoNTA-ZFP showed impaired exercise performance in the balance beam experiment compared to buffered saline (FIG. 9).

[0112] Example 9: Footprint Test In this example, an alternative method for measuring the muscle paralyzing effects of drugs is examined.

[0113] method BoNTA-ZFP was applied topically to CJ57 female mice as described in Example 4. Mice were subjected to footprint analysis experiments. The footprint width was recorded.

[0114] Mice treated with Botox injections or topical BoNTA-ZFP showed reduced contact area in paw print analysis compared to buffered saline (FIG. 10).

[0115] Examples 8-10 demonstrate the validity and quantitative nature of these tests for measuring the muscle paralyzing effects of test agents.

[0116] The present invention is not limited in scope by the specific embodiments described, which are intended as single descriptions of individual aspects of the invention, and compositions or methods that are functionally equivalent are within the scope of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present invention without departing from the spirit or scope of the invention. Such modifications are intended to fall within the scope of the appended claims. Thus, the present invention is intended to cover the modifications and variations of the present invention provided they come within the scope of the appended claims and their equivalents.

[0117] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A chimeric polypeptide comprising: (a) a botulinum toxin (BoNT) light chain; (b) a BoNT heavy chain; and (c) a transcriptional activator of transcription (TAT) or a pep-1 peptide (Pep-1), wherein the TAT or Pep-1 is located at the N-terminus of the BoNT light chain, the BoNT light chain is located at the N-terminus of the BoNT heavy chain, or the BoNT heavy chain is located at the N-terminus of the BoNT heavy chain, and the BoNT heavy chain is linked to the BoNT light chain via a disulfide bond.

2. The chimeric polypeptide of claim 1 comprising no more than 2000 amino acid residues.

3. The chimeric polypeptide of claim 1 or 2, wherein the BoNT light chain and the TAT or Pep-1 are on the same peptide chain.

4. The chimeric polypeptide of claim 1 or 2, wherein the BoNT light chain and the TAT or Pep-1 are on different peptide chains.

5. 5. The chimeric polypeptide of any one of claims 1 to 4, wherein the BoNT is selected from BoNT A, B, C, D, E, F, or G, or a variant thereof having at least 90% sequence identity thereto.

6. The chimeric polypeptide of claim 5, wherein the BoNT is selected from BoNT subtypes A1 to A10, B1 to B8, E1 to E9, and F1 to F7.

7. The chimeric polypeptide of claim 5 , wherein the BoNT is BoNT A.

8. The chimeric polypeptide of any one of claims 1 to 7, wherein the BoNT light chain comprises the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

9. The chimeric polypeptide of any one of claims 1 to 8, wherein the BoNT heavy chain comprises the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

9.

10. The chimeric polypeptide of claim 1 or 3, comprising a single-chain polypeptide comprising the amino acid sequence of SEQ ID NO:

16.

11. The chimeric polypeptide of claim 1 or 3, comprising a single-chain polypeptide comprising the amino acid sequence of SEQ ID NO:

17.

12. A formulation comprising the chimeric polypeptide of any one of claims 1 to 11 for topical or intramuscular application.

13. 13. The formulation of claim 12, wherein the topical application is to the skin or to the mucous membranes of the eye, ear, nose, mouth, lips, urethral meatus, anus, or tongue.

14. 13. The formulation of claim 12, wherein the topical application is to a disrupted stratum corneum of skin.

15. 15. The formulation of claim 14, wherein the disruption is performed using a needle or microneedle used to deliver the formulation.

16. The formulation of any one of claims 12 to 15, wherein the topical application is to the skin of the face or neck of a mammal.

17. The formulation of any one of claims 12 to 15, wherein the formulation comprises a cream, gel, or spray.

18. The formulation of any one of claims 12 to 15, wherein the application is to a mammal in need of treatment for facial wrinkles, dystonia, spasticity, hemifacial spasm, hyperhidrosis, or hypersalivation.

19. A polynucleotide encoding the chimeric polypeptide according to any one of claims 1 to 11.

20. A cell comprising the polynucleotide of claim 19.

21. 21. The cell of claim 20 which is a bacterial cell.

22. 22. The cell of claim 21 , wherein the bacterial cell is a Clostridium botulinum cell.

23. 21. The cell of claim 20 which is an insect cell.

24. The cell of claim 23 , wherein the insect cell is a Spodoptera cell.

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