Sequence modification of recombinant botulinum neurotoxin type a2 and use thereof

Abstract

Provided are modified botulinum neurotoxin type A2, and a preparation method therefor and a use thereof. The modified botulinum neurotoxin type A2 comprises an LC domain and an HN domain, wherein a structural unit enabling the LC domain and the HN domain to undergo controllable cleavage under a predetermined condition is artificially set between the LC domain and the HN domain; optionally, one or more specific protease cleavage sites are comprised between the last cysteine at the C‑terminus of the LC domain and the first cysteine at the N‑terminus of the HN domain.

Description

Sequence Modifications and Applications of Recombinant A2 Botulinum Toxin

[0001] Cross-referencing

[0002] This disclosure claims priority to PCT application PCT / CN2024 / 137446, filed on December 6, 2024, the contents of which are considered part of this disclosure and are incorporated herein in their entirety. Technical Field

[0003] This disclosure generally relates to the field of recombinant protein technology, and specifically to modified A2 botulinum toxin, its recombinant production, and its uses. Background Technology

[0004] Botulinum neurotoxins (BoNTs) are among the most potent known protein toxins (Dong et al., Annu Rev. Biochem, 2019, 88:811-837). Members of the natural botulinum toxin family can be produced by several bacteria belonging to the genus Clostridium, primarily including various types of Clostridium botulinum, as well as other natural strains containing the botulinum toxin gene, such as Clostridium baratii, Clostridium butyricum, Clostridium sporogenes, and Clostridium argentinense.

[0005] The botulinum toxin family currently includes seven major serotypes (BoNT / AG). Serum typing based on seroreactivity is a traditional method: antibodies produced after immunizing an animal with one serotype generally cannot cross-neutralize different serotypes. This is because there are significant differences in protein sequences between different serotypes (up to 70%). All members of the botulinum toxin family are multi-domain proteins of approximately 150 kDa, possessing similar three-dimensional protein structures and sharing the same mechanism of action and function. The botulinum toxin protein consists of a light chain (LC, ~50 kDa, which is a protease domain) and a heavy chain (HC, ~100 kDa). The heavy chain can be further divided into a transmembrane transport domain (H). N (~50kDa), and a receptor-binding domain (H C (~50kDa). Botulinum toxin specifically targets nerve endings through its receptor-binding domain, then delivers light chains into nerve cells via its transmembrane transport domain. Finally, the protease activity of the toxin light chains cleaves key protein substrates within the nerve, thereby blocking the release of neurotransmitters and achieving muscle paralysis.

[0006] Differences in protein sequences between different serotypes can lead to their targeting of different cell surface receptors and cleavage of different neuronal protein substrates. For example, BoNT / A recognizes the SV2 receptor, while BoNT / B recognizes synaptotagmin I / II, both of which can target neurons. Furthermore, BoNT / A, E, and C cleave the neuronal protein SNAP-25, while BoNT / B, D, F, and G cleave another protein, VAMP1 / 2. Both groups of proteins are essential for neurotransmitter release, so cleavage of either protein can block neurotransmitter release. In addition to these functionally specific differences, different protein sequences can also cause significant differences in pharmacokinetics, such as in vivo stability, onset time, and duration of action.

[0007] In recent years, it has been discovered that each serotype can be further subdivided into multiple subtypes (Peck et al., Toxins, 2017, 9:38). For example, botulinum toxin type A can be divided into eight subtypes: BoNT / A1-A8. Different subtypes are produced by different Clostridium botulinum strains. The protein sequences of these subtypes can differ significantly; for example, the protein sequence differences between botulinum toxin type A subtypes range from 2.6% to 15.4% (Figure 1). While the protein sequence differences between subtypes generally do not alter their specificity for receptors and enzyme substrates as much as serotypes do, they can cause differences in protein expression levels, protein stability, protein activity, in vivo toxicity, immunogenicity, and in vivo pharmacokinetics. This is especially true for subtypes with relatively large protein differences.

[0008] For example, the protein sequences of A1 and A2 types show a significant overall difference of 10.1% (Figure 1). According to literature reports, A2 type exhibits a faster onset of action than A1 type (Pier et al., FEBS Lett., 2011, 585:199-206). A2 type also shows significantly different immunogenicity compared to A1 type: literature reports that antibodies against A1 cannot completely neutralize A2 toxins (Kozaki et al., Microbiol. Immunol., 1995, 39:767-774; Sakaguchi et al., Int. J. Food Microbiol., 1990, 11:231-242). This finding strongly demonstrates that the surface amino acids of A1 and A2 are very different. They also differ significantly in their protein biochemical characteristics; for example, the heavy chain molecular weight of A2 is 101 kDa, while that of A1 is 93 kDa. Literature also reports differences in their sensitivity to exogenous proteases. Specifically, of the 847 amino acids in the heavy chain, A2 and A1 differ by as many as 109 amino acids, a proportion reaching 13% (Cordoba et al., System Appl. Microbiol., 1995, 18:13-22). Therefore, A2 and A1 botulinum toxins, besides sharing a common mechanism of action and target, differ significantly in protein sequence, biochemical characteristics, and immunogenicity. From the perspective of protein drug development, they can be considered as two different protein drugs targeting the same target.

[0009] Because botulinum toxin can block nerve activity, this effect can be used to treat various neurological disorders and also to eliminate wrinkles in cosmetic medicine (Johnson EA., Annu. Rev. Microbiol., 1999, 53: 551-575). Currently marketed type A botulinum toxin products are all based on the exact same A1 natural sequence (UniprotKB: P0DPI1). This toxin was approved for marketing more than 30 years ago and has been widely used for over 30 years. Current annual sales reach several billion US dollars.

[0010] Each different natural toxin sequence originates from a different strain. However, strains suitable for industrial production must meet a series of conditions and are not easily found. The A1 type toxin in marketed drugs comes from an early-discovered superior strain called Hall (or a strain very closely related to Hall). Natural botulinum toxin is first transcribed into a complete 150kDa protein in Clostridium botulinum, which also produces several accessory proteins. These accessory proteins bind to the toxin protein, forming a large toxin complex (e.g., 900kDa). Currently, the main products on the market (such as Botox) are purified 900kDa toxin complexes. Because the primary function of these accessory proteins is to protect botulinum toxin for absorption in the intestines of animals and humans, their usefulness is limited in injectable applications. New products further purify the 900kDa toxin complex to obtain the botulinum toxin protein itself, resulting in a final product containing only 150kDa toxin protein. Product development and clinical experience have shown that the lack of accessory protein protection does not affect the activity of A1.

[0011] A key aspect of botulinum toxin activity is its activation via protease cleavage. This is because although botulinum toxin initially transcribes into a precursor protein, it only becomes active after breaking down into two fragments: a C-terminal light chain and an N-terminal heavy chain. The junction between the light and heavy chains needs to be cleaved by a protease. The separated light and heavy chains remain linked by a disulfide bond. Functionally, the heavy chain targets nerve cells and then transports the toxin light chain across the membrane into the cell. Once the light chain reaches the cell, the disulfide bond is broken, releasing the light chain into the cell. Therefore, an unactivated toxin molecule cannot release the light chain into the cell.

[0012] The Hall strain currently used in industrial production not only has a relatively high toxin yield but also expresses the necessary proteases to activate botulinum toxin. Protease cleavage occurs on the 900 kDa toxin complex, released into the culture medium after bacterial lysis. The presence of accessory proteins ensures that the toxin protein is cleaved only at the junction of the light and heavy chains, while other parts are not further cleaved, thus maintaining the integrity and full activity of the toxin protein. The toxin protein extracted from the Hall strain is fully activated, which is a significant advantage of this strain. Different strains have varying protease expression capabilities; some natural strains even lack the necessary proteases and cannot fully activate the toxin protein.

[0013] One disadvantage of using natural Clostridium botulinum strains to produce botulinum toxin is biosafety concerns. Natural Clostridium botulinum produces a botulinum toxin complex, which has high oral toxicity. If maliciously added to food, it can cause poisoning. Therefore, natural strains capable of producing botulinum toxin are subject to strict control by various countries. The entire production process, because it produces a highly oral toxic botulinum toxin complex, also requires strict control, resulting in high production costs.

[0014] In recent years, due to limitations in using natural strains, there is a market demand for new methods to produce botulinum toxin. Several laboratories and companies are exploring recombinant protein methods to produce A1 type botulinum toxin. Common recombinant protein expression systems include *E. coli*, *Bacillus subtilis*, yeast, and insect cells. One advantage of recombinant expression is that it can express only 150 kDa botulinum toxin protein without producing other accessory proteins. Without the protection of accessory proteins, botulinum toxin itself can be degraded in the digestive tract, thus significantly reducing oral toxicity and lowering biosafety concerns and regulatory requirements. Furthermore, recombinant expression can overcome the dependence on screening for suitable *Clostridium botulinum* strains for production.

[0015] However, recombinant protein production also presents many unknowns that need to be explored and resolved. One major unknown is whether the expressed and purified botulinum toxin protein can achieve the same activity and toxicity as the toxin expressed from natural Clostridium botulinum. For botulinum toxin protein drug development, protein activity and toxicity are crucial indicators. This is because poor activity necessitates higher injection doses, and since botulinum toxin is an exogenous protein, high injection doses significantly increase the likelihood of the body producing neutralizing antibodies. It is currently unclear whether the correct folding of botulinum toxin requires a Clostridium botulinum-specific environment, or whether high botulinum toxin activity requires specific post-transcriptional modifications specific to the Clostridium botulinum environment. Furthermore, different expression hosts express different endogenous proteases, which may cleave the botulinum toxin protein at different sites, reducing protein expression and activity. These conditions may differ for each different botulinum toxin serotype and subtype. For example, sequence variations may affect protein folding and sensitivity to host proteases. Therefore, it is theoretically and technically possible to produce different botulinum toxin serotypes and subtypes using recombinant methods simply by expressing and purifying the protein. However, it is difficult to predict whether the expressed toxin protein can achieve the high activity level of the natural toxin purified from Clostridium botulinum based on current knowledge.

[0016] Currently published literature reports the successful production of highly active recombinant type A botulinum toxin, all of which are derived from the already marketed type A1 botulinum toxin product (UniprotKB:P0DPI1). These reports demonstrate that A1 can be produced recombinantly while maintaining high activity. However, whether other subtypes of type A toxin (A2-A8) can be produced recombinantly and achieve the same high activity as their corresponding natural toxins remains unknown and unpredictable. Only one existing publication reports the recombinant production of type A2 botulinum toxin using *E. coli* and the testing of the purified toxin's activity (Kohda et al, *Microbiology and Immunology*, 2020, 64:502-511). Other subtypes have not yet been reported. Data from this publication shows that the activity of the type A2 toxin produced using *E. coli* in mice was only one-tenth that of the natural type A2 purified from *Botulinum toxin*. This publication demonstrates that whether type A2 can achieve the activity level of the natural toxin through recombinant production is unpredictable and difficult to achieve. Summary of the Invention

[0017] Because A2 and A1 differ significantly in protein sequence, biochemical characteristics, immunogenicity, and potential pharmacokinetics, A2 botulinum toxin has the potential to be further developed into superior new drugs. However, these differences also mean that developing A2 into a drug requires starting from scratch, necessitating extensive exploration and experimental validation, and carrying a significant risk of drug development failure. Perhaps considering these unpredictable risks, only A1 botulinum toxin products have been developed for the market. Natural Clostridium botulinum capable of producing A2 is strictly controlled and difficult to obtain. Therefore, there is a strong market demand for new technologies that can recombinantly produce A2 botulinum toxin with the same high activity as the natural toxin. Due to the various protein differences between A2 and A1, the recombinant expression of A2 requires extensive exploration and validation, especially considering the need to maintain the high activity of the A2 protein obtained from recombinant expression, rather than simply obtaining the protein itself. Currently, there are no successful cases reported in the published literature. Furthermore, even if it can be produced and purified using recombinant production methods, whether A2 can be manufactured into a stable drug remains unknown.

[0018] This disclosure discloses a sequence modification of A2 toxin by adding a specific protease cleavage site, and successfully demonstrated that recombinant expression of the modified A2 toxin can be achieved using *E. coli* as the expression host, and that after activation with a specific protease, the activity reaches the same level as natural A2 toxin. Furthermore, this invention has successfully developed a drug with A2 toxin as the active ingredient and verified that it exhibits superior effects compared to A1 botulinum toxin.

[0019] In a first aspect, this disclosure provides a modified type A2 botulinum toxin, wherein the modified type A2 botulinum toxin comprises an LC domain and an HN domain, and structural units are artificially provided between the LC domain and the HN domain such that the LC domain and the HN domain can be controllably broken under predetermined conditions.

[0020] Optionally, a structural unit is artificially provided between the last cysteine ​​residue at the C-terminus of the LC domain and the first cysteine ​​residue at the N-terminus of the HN domain, such that the LC domain and the HN domain can be controllably broken under predetermined conditions.

[0021] Optionally, one or more specific protease cleavage sites are contained between the last cysteine ​​residue at the C-terminus of the LC domain and the first cysteine ​​residue at the N-terminus of the HN domain.

[0022] Optionally, the modified A2 botulinum toxin contains one or more specific protease cleavage sites between cysteine ​​residues 430 and 454, wherein the site is defined by the amino acid sequences of NCBI ID:WP_012703873.1 (SEQ ID NO:87), NCBI ID:ABC26002 (SEQ ID NO:102), NCBI ID:ADB85243 (SEQ ID NO:98), NCBI ID:ADN04908 (SEQ ID NO:105), NCBI ID:AFM77673.1 (SEQ ID NO:100), NCBI ID:AFV13855.1 (SEQ ID NO:99), NCBI ID:AIT75847.1 (SEQ ID NO:97), and NCBI ID:AJE13255 (SEQ ID NO:104). The amino acid sequences of NCBI ID: KEI94032.1 (SEQ ID NO: 101), NCBI ID: KEI94982 (SEQ ID NO: 103), NCBI ID: NFD83166.1 (SEQ ID NO: 110), NCBI ID: NFJ22948.1 (SEQ ID NO: 107), NCBI ID: WP_079007110 (SEQ ID NO: 106), NCBI ID: WP_205607627 (SEQ ID NO: 109), and / or NCBI ID: WP_228605784 (SEQ ID NO: 108) are used as references.

[0023] The modified A2 botulinum toxin disclosed herein can be cleaved by specific proteases, achieving an activity level consistent with that of natural A2 toxin.

[0024] Secondly, this disclosure provides a nucleic acid molecule encoding the modified A2 type botulinum toxin of the first aspect of this disclosure.

[0025] Thirdly, this disclosure provides an expression vector containing the nucleic acid molecule described in the second aspect of this disclosure.

[0026] Fourthly, this disclosure provides a host cell comprising a nucleic acid molecule of the second aspect of this disclosure or an expression vector of the third aspect of this disclosure;

[0027] The host cells were selected from Escherichia coli cells, Bacillus subtilis cells, yeast cells, insect cells, and mammalian cells;

[0028] Optionally, the host cell is Escherichia coli.

[0029] Fifthly, this disclosure provides a method for preparing modified A2 botulinum toxin, comprising culturing host cells according to the fourth aspect of this disclosure under conditions favorable to the expression of the modified A2 botulinum toxin of the first aspect of this disclosure.

[0030] Sixthly, this disclosure provides a method for producing soluble double-stranded type A2 botulinum toxin, the method comprising:

[0031] Step (1): Contact the modified A2 botulinum toxin of the first aspect of this disclosure or the modified A2 botulinum toxin prepared by the preparation method of the fifth aspect of this disclosure with a specific protease capable of enzymatically cleaving specific protease cleavage sites.

[0032] In a seventh aspect, this disclosure provides a composition comprising soluble double-stranded type A2 botulinum toxin prepared using the preparation method of the sixth aspect of this disclosure.

[0033] Eighthly, this disclosure provides the use of the modified A2 botulinum toxin of the first aspect of this disclosure, the modified A2 botulinum toxin prepared by the preparation method of the fifth aspect of this disclosure, the soluble double-chain A2 botulinum toxin prepared by the preparation method of the sixth aspect of this disclosure, or the composition of the seventh aspect of this disclosure in the preparation of cosmetic products.

[0034] Ninthly, this disclosure provides the use of the modified A2 botulinum toxin of the first aspect of this disclosure, the modified A2 botulinum toxin prepared by the preparation method of the fifth aspect of this disclosure, the soluble double-stranded A2 botulinum toxin prepared by the preparation method of the sixth aspect of this disclosure, or the composition of the seventh aspect of this disclosure in the preparation of a medicament for reducing neuronal activity.

[0035] In a tenth aspect, this disclosure provides a method for treating, preventing, or alleviating a disease, symptom, or disorder, the method comprising: administering to a subject in need a therapeutically effective amount of a modified A2 botulinum toxin of the first aspect of this disclosure, a modified A2 botulinum toxin prepared using the preparation method of the fifth aspect of this disclosure, a soluble double-stranded A2 botulinum toxin prepared using the preparation method of the sixth aspect of this disclosure, or a composition of the seventh aspect of this disclosure. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 shows a comparison of protein sequence differences among currently known botulinum toxin type A subtypes.

[0038] Figure 2 shows the three domains of natural A2 botulinum toxin (light chain Lc, transmembrane transport Hn, receptor binding Hc) and the corresponding recombinant A2 botulinum toxin; a specific protease recognition site is inserted between the light chain (Lc) and the heavy chain of the recombinant A2 botulinum toxin.

[0039] Figure 3 shows the recombinant A2 toxin (A2-4) protein products after purification and thrombin activation in two batches in Example 2. The purity was analyzed by protein gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining.

[0040] Figure 4 shows the recombinant A2 toxin (A2-4) protein products from two batches in Example 2 after purification and thrombin activation, further analyzed using the commonly used size exclusion chromatography (SEC-HPLC) method. The samples showed a single main peak in the SEC-HPLC detection. The purities of the two batches were 99.4% and 99.1%, respectively.

[0041] Figure 5 shows the comparison of DAS scores induced by recombinant A2 type (A2-4) and natural A1 type toxins in Example 4 using the mouse behavioral hind toe abduction score (DAS). Scores range from 0 to 4, with 4 representing complete muscle paralysis and no hind toe abduction. The solvent group represents the control (only injected with the toxin-free reconstituted solution). 1 U represents one mouse LD50. 50 .

[0042] Figure 6 shows the comparison of mouse weight loss induced by recombinant A2 type (A2-4) toxin and natural A1 type toxin in Example 4 using the mouse behavioral hind toe abduction score (DAS) experiment.

[0043] Figure 7 shows the comparison of muscle strength decreases in mice induced by recombinant A2 type (A2-4) toxin and natural A1 type toxin in Example 5, measured using electrophysiological methods.

[0044] Figure 8 shows the SDS-PAGE spectra of the non-reduced and reduced samples of A2-1, A2-2, and A2-3 after thrombin digestion in Example 6. The seven lanes in Figure 8 are, in order, the Marker, the non-reduced and reduced samples of A2-1 after thrombin treatment, the non-reduced and reduced samples of A2-2 after thrombin treatment, and the non-reduced and reduced samples of A2-3 after thrombin treatment.

[0045] Figure 9 shows the DAS scores of mice A2-1, A2-2, and A2-3 in Example 6. Figure 9(A) is a graph showing the change of DAS score of A2-1 over time; Figure 9(B) is a graph showing the change of DAS score of A2-2 over time; Figure 9(C) is a graph showing the change of DAS score of A2-3 over time. In the figures, the Y-axis represents the average DAS score of each group of mice, and the X-axis represents the number of days after the injection of botulinum neurotoxin.

[0046] Figure 10 shows the SDS-PAGE spectra of the following samples from Example 7: A2-1 after thrombin digestion (both non-reduced and reduced), A2-TEV after TEV enzyme digestion (both non-reduced and reduced), A2-3C after 3C protease digestion (both non-reduced and reduced), natural A2 botulinum toxin protein after thrombin digestion (both non-reduced and reduced), and natural A2 botulinum toxin protein after trypsin digestion (both non-reduced and reduced). The 11 lanes in Figure 10 represent, in order: Marker, A2-1 after thrombin treatment (both non-reduced and reduced), A2-TEV after TEV enzyme digestion (both non-reduced and reduced), A2-3C after 3C protease digestion (both non-reduced and reduced), natural A2 botulinum toxin protein after thrombin digestion (both non-reduced and reduced), and natural A2 botulinum toxin protein after trypsin digestion (both non-reduced and reduced).

[0047] Figure 11 shows the DAS scores of A2-1, A2-3C, and A2-TEV in Example 7. Figure 11(A) is a graph showing the change of DAS score of A2-1 over time; Figure 11(B) is a graph showing the change of DAS score of A2-3C over time; Figure 11(C) is a graph showing the change of DAS score of A2-TEV over time.

[0048] Figure 12 shows the SDS-PAGE spectra of the non-reduced and reduced samples of A2-1, A2-P2, and A2-L2 after thrombin digestion in Example 8. The seven lanes in Figure 12 are, in order, the Marker, the non-reduced and reduced samples of A2-1 after thrombin treatment, the non-reduced and reduced samples of A2-P2 after thrombin treatment, and the non-reduced and reduced samples of A2-L2 after thrombin treatment.

[0049] Figure 13 shows the DAS scores of A2-1, A2-P2, and A2-L2 in Example 8. Figure 13(A) is a graph showing the change of DAS score of A2-1 over time; Figure 13(B) is a graph showing the change of DAS score of A2-P2 over time; Figure 13(C) is a graph showing the change of DAS score of A2-L2 over time. Detailed Implementation

[0050] (I) Definitions or terms

[0051] The following abbreviations are used to refer to the definitions and terms used in this disclosure. Unless otherwise defined, all technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. The following terms are provided below.

[0052] In this document, the singular forms “a / an” and “the” include the plural forms unless the context explicitly indicates otherwise. As will be apparent to those skilled in the art from the teachings contained herein, when used in the context of numerical values ​​and ranges, the term “about” or “approximately” means a value or range that is approximately or close to the specified value or range, such that the embodiment can be performed as intended. In some embodiments, “about” means a numerical amount ±10%.

[0053] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length. They may include one or more ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, such as locked nucleic acids (LNAs) and peptide nucleic acids (PNAs).

[0054] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. Proteins or peptides typically contain at least two amino acids or amino acid variants, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids or variants linked together by peptide bonds. These terms include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0055] The terms “identity” and “homology” are used interchangeably in this document and, when used to describe polynucleotide or polypeptide sequences, refer to the percentage of identical bases or amino acids in the same relative positions when comparing or aligning two sequences of a polypeptide or polynucleotide. Sequence identity can be determined in a variety of different ways. For example, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.).

[0056] The nucleic acid molecule described in this disclosure can be, for example, an isolated nucleic acid molecule. The term "isolated," when used for a nucleic acid molecule or protein molecule, refers to a nucleic acid or protein that has been separated from one or more components associated with it that are normally present in the natural environment. Separation can include detachment from a larger nucleic acid (e.g., from a gene or chromosome) or from other proteins or molecules that are normally in contact with said nucleic acid or protein. The term covers, but does not require, complete separation.

[0057] The nucleic acid molecules provided in this disclosure can be of any type, as long as they can achieve the purpose of this disclosure. For example, the nucleic acid molecules of this disclosure can be single-stranded or double-stranded deoxyribonucleic acid molecules (i.e., DNA molecules) or single-stranded or double-stranded ribonucleic acid molecules (i.e., RNA molecules); the nucleic acid molecules of this disclosure can be linear or circular.

[0058] The term "host cell" as used in this disclosure refers to a eukaryotic or prokaryotic cell into which one or more deoxyribonucleic acid (DNA) or vectors have been introduced. It should be understood to refer not only to the specific target cell but also to its offspring or potential offspring. Although offspring may differ from their parent cells due to mutations or environmental influences, they are still included within the scope of the terminology used in this specification.

[0059] The terms "botulinum toxin," "botulinum toxin," and "neurotoxin" used in this disclosure are general terms referring to botulinum toxin polypeptides that enter neurons and inhibit the release of neurotransmitters. This process encompasses the binding of the neurotoxin to low- or high-affinity receptors, the internalization of the neurotoxin, the transmembrane transport of the neurotoxin's endopeptidase portion into the cytoplasm, and the enzymatic cleavage of the substrate by the neurotoxin. More specifically, the term "neurotoxin" encompasses any botulinum toxin polypeptide produced by Clostridium bacteria (clostridium neurotoxin) that enters neurons and inhibits the release of neurotransmitters, as well as such botulinum toxin polypeptides produced through recombinant or chemical techniques. The double-stranded form is the active form of botulinum toxin.

[0060] The terms "modification," "transformation," "sequence modification," and "protein sequence modification" used in this article are general terms referring to altering the protein sequence of natural botulinum toxin by inserting new sequences or deleting parts of the sequence. "Natural" and "natural toxin" are general terms referring to botulinum toxin protein purified from Clostridium botulinum, whose protein sequence has not undergone any artificial modification. "Recombinant" and "recombinant toxin" refer to botulinum toxin protein expressed and purified in common protein expression systems such as Escherichia coli, Bacillus subtilis, yeast, and insect cells.

[0061] The "activity" or "bioactivity" of botulinum toxin refers to its ability to block nerve signal transmission at synapses containing acetylcholine receptors by blocking the release of acetylcholine from nerve endings. This term is interchangeable with the terms "toxicity," "in vivo toxicity," "in vivo activity," "inhibition of cholinergic transmission," "inhibition of cholinergic input," "reduction of cholinergic input," and their tendencies. The in vitro assays of the botulinum toxin bioactivity described herein can be performed using common mouse median lethal doses (LD50). 50 The method for detection is as follows. In this experiment, the "unit" (U) of activity is defined as the amount of toxin protein required to kill 50% of the test mice within four days after injection (1 LD). 50 (1U).

[0062] The in vivo efficacy of neurotoxins can be determined using the hind toe abduction score (DAS) (a measure of muscle paralysis). The neurotoxin was injected into the gastrocnemius / soleus muscle complex of mice, and the hind toe abduction score was then assessed using Aoki's method (Aoki KR, Toxicon, 2001, 39: 1815-1820) to perform the DAS measurement. In the DAS measurement, mice were briefly suspended by their tails to elicit a characteristic panic response, in which the mice extended their hind limbs and abducted their hind toes. Following neurotoxin injection, varying degrees of hind toe abduction were scored on a five-point scale (0 = normal to 4 = maximum reduction in hind toe abduction and leg extension).

[0063] As used herein, the term "conservative substitution" refers to an amino acid change that results in the replacement of one amino acid with a chemically similar amino acid. For example, one acidic amino acid may be replaced by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another neutral amino acid. Tables of conserved substitutions of functionally similar amino acids are well known in the art. Amino acid modifications, such as substitutions, can be introduced into the botulinum toxin sequences of this disclosure using standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. The term "conservative substitution" also refers to the substitution of one or more amino acids in a polypeptide without substantially losing its functionality. One amino acid can be substituted for another without losing the activity of the polypeptide. For example, the following amino acids can be interchanged:

[0064] Ala, Ser, Thr, Gly (small aliphatic, nonpolar, or micropolar residues);

[0065] Asp, Asn, Glu, Gln (polar, negatively charged residues and their amides);

[0066] His, Arg, Lys (polar, positively charged residues);

[0067] Met, Leu, Ile, Val (Cys) (large aliphatic, nonpolar residues);

[0068] Phe, Tyr, Trp (large aromatic residues).

[0069] Optional "substitutions" are those that are conservative, meaning that the residues are substituted by another of the same general types. When making changes, the hydrophobicity index of the amino acid can be taken into account. Some amino acids can be substituted by other amino acids with similar hydrophobicity indices or scores and still produce botulinum toxins with similar biological activities. When making such changes, substitution of amino acids with a hydrophobicity index within ±2 is preferred, within ±1 is more preferred, and within ±0.5 is even more preferred. Similarly, the selected amino acid can be substituted by other amino acids with similar hydrophilicity. When making such changes, as with the hydrophobicity index, substitution of amino acids with a hydrophobicity index within ±2 is preferred, within ±1 is more preferred, and within ±0.5 is even more preferred.

[0070] An example replacement is shown below:

[0071] The table above lists amino acids represented by one or three letter abbreviations. The amino acids are: alanine (A, Ala), cysteine ​​(C, Cys), aspartic acid (D, Asp), glutamic acid (E, Glu), phenylalanine (F, Phe), glycine (G, Gly), histidine (H, His), isoleucine (I, Ile), lysine (K, Lys), leucine (L, Leu), methionine (M, Met), asparagine (N, Asn), proline (P, Pro), glutamine (Q, Gln), arginine (R, Arg), serine (S, Ser), threonine (T, Thr), valine (V, Val), tryptophan (W, Trp), and tyrosine (Y, Tyr).

[0072] As used herein, the term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or disease progression in the treated individual. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or remission are used, they also imply elimination, disappearance, or non-occurrence.

[0073] (II) Specific Implementation Methods

[0074] Modified type A2 botulinum toxin

[0075] In a first aspect, this disclosure provides a type A2 botulinum toxin, wherein the modified type A2 botulinum toxin comprises L C Domain and H N Structural domain, the L C Structural domain and the H N The artificial arrangement between structural domains makes the L C Structural domain and the H N A structural unit whose structural domain can be controlled to fracture under predetermined conditions.

[0076] When used herein, the term "fractured structural unit" refers to any structural unit that can be fractured under predetermined conditions. The structural unit can be a polypeptide or a chemical structure. The structural unit can be artificially inserted into the modified A2 botulinum toxin through gene editing and recombination, or artificially inserted into the modified A2 botulinum toxin through other operable means.

[0077] In some embodiments, the predetermined conditions are conditions or operations that can cause the structural unit to break. In some specific embodiments, the structural unit is temperature-sensitive, and the predetermined condition is a set temperature that can cause the structural unit to break. In some specific embodiments, the structural unit is pH-sensitive, and the predetermined condition is a set pH value that can cause the structural unit to break. In some specific embodiments, the structural unit is enzyme-sensitive, and the predetermined condition is contacting the modified A2 type botulinum toxin with an enzyme that can cause the structural unit to break.

[0078] In some implementations, the L C The last cysteine ​​residue at the C-terminus of the domain and the H N The L domain is artificially positioned between the first cysteine ​​residue at the N-terminus. C Structural domain and the H N A structural unit whose structural domain can be controlled to fracture under predetermined conditions.

[0079] In some implementations, the L C The last cysteine ​​residue at the C-terminus of the domain and the H N The first cysteine ​​residue at the N-terminus of the domain contains one or more specific protease cleavage sites.

[0080] In some embodiments, the modified A2 type botulinum toxin comprises H C Structural domain.

[0081] When used in this document, the term "L" C The "domain" refers to the protease domain of botulinum toxin protein, which has zinc-dependent protease activity and is the catalytic active center of the toxin.

[0082] When used in this document, the term "H" N The "transmembrane transport domain" refers to the transmembrane transport domain of the botulinum toxin protein, which is responsible for transporting L... C The structural domain is transported into the neuronal cell.

[0083] When used in this document, the term "H" C The "receptor-binding domain" refers to the receptor-binding domain of the botulinum toxin protein, which binds to receptors on the surface of neurons, allowing the toxin to enter the neurons and exert its toxic effects.

[0084] When used in this document, the term "L" C "The last cysteine ​​residue at the C-terminus of the domain" refers to the residue in the Lc domain that is C-terminus of the Lc domain. C The N-terminus of the structural domain to the L-terminus C The C-terminus of the structural domain is sequential, and the distance L is... C The nearest cysteine ​​residue to the C-terminus of the domain.

[0085] When used in this document, the term "H" N The "first cysteine ​​residue at the N-terminus of the domain" refers to the residue at the H-terminus. N In the structural domain, with H N The N-terminus of the structural domain to the H-terminus N The C-terminus of the structural domain is sequential, and the distance L is... N The nearest cysteine ​​residue at the N-terminus of the domain.

[0086] In some embodiments, novel recombinant botulinum neurotoxin L is arbitrarily inserted into the protease cleavage site. C The last cysteine ​​residue and H at the C-terminus of the domain N Between the first cysteine ​​residue at the N-terminus of the domain.

[0087] In some implementations, the novel recombinant botulinum neurotoxin L C The last cysteine ​​residue and H at the C-terminus of the domain N The amino acid sequence between the first cysteine ​​residue at the N-terminus of the domain is replaced by a protease cleavage site. In some embodiments, the novel recombinant botulinum neurotoxin L... C The last cysteine ​​residue and H at the C-terminus of the domain N An amino acid between the first cysteine ​​residue at the N-terminus of the domain is replaced by a protease cleavage site. In some embodiments, the novel recombinant botulinum neurotoxin L... C The last cysteine ​​residue and H at the C-terminus of the domain N Two or more consecutive amino acids between the first cysteine ​​residue at the N-terminus of the domain are replaced by a protease cleavage site.

[0088] In some embodiments, the modified A2 botulinum toxin contains one or more specific protease cleavage sites between cysteine ​​residues 430 and 454, wherein the site is defined by the amino acid sequences of NCBI ID:WP_012703873.1 (SEQ ID NO:87), NCBI ID:ABC26002 (SEQ ID NO:102), NCBI ID:ADB85243 (SEQ ID NO:98), NCBI ID:ADN04908 (SEQ ID NO:105), NCBI ID:AFM77673.1 (SEQ ID NO:100), NCBI ID:AFV13855.1 (SEQ ID NO:99), NCBI ID:AIT75847.1 (SEQ ID NO:97), and NCBI ID:AJE13255 (SEQ ID NO:104). The amino acid sequences of NCBI ID: KEI94032.1 (SEQ ID NO: 101), NCBI ID: KEI94982 (SEQ ID NO: 103), NCBI ID: NFD83166.1 (SEQ ID NO: 110), NCBI ID: NFJ22948.1 (SEQ ID NO: 107), NCBI ID: WP_079007110 (SEQ ID NO: 106), NCBI ID: WP_205607627 (SEQ ID NO: 109), and / or NCBI ID: WP_228605784 (SEQ ID NO: 108) are used as references.

[0089] Enzymatic activation of botulinum toxin is a specific post-transcriptional modification unique to natural Clostridium botulinum and a crucial step for botulinum toxin activity. Recombinant botulinum toxin production requires designing new methods to address this activation issue. Several approaches have been reported in the literature, including using proteases with relatively low specificity, such as endoproteinase Lys-C and trypsin (see patent CN 106414759 B; Kohda et al, Microbiology and Immunology, 2020, 64:502-511). The advantage of using these proteases is that they do not require modification of the natural toxin sequence. The disadvantage is their low specificity; they cleave multiple sites, requiring the establishment of strict incubation conditions to avoid toxin protein degradation and making it difficult to maintain stable quality. Another common approach is to insert new specific protease recognition and cleavage sites into the region connecting the light and heavy chains, thus allowing the use of more specific proteases. This method can significantly reduce (but not completely eliminate) the probability of non-specific cleavage of other parts of the toxin. However, this method requires introducing new protein sequences into the light and heavy chain linking regions of the toxin. This method of inserting specific protease cleavage sites has been reported for A1 (e.g., Wang et al., J. Biol. Chem., 2011, 286:6375-85), but its application to A2 toxin has not yet been publicly reported.

[0090] This disclosure provides a sequence-modified A2 toxin containing a specific protease cleavage site introduced in the middle of the linker sequence between the light and heavy chains. This allows for the use of a highly specific exogenous protease to cleave the linker sequence after recombinant expression, thereby activating the A2 toxin (Figure 2).

[0091] In some embodiments, the specific protease cleavage site is selected from: thrombin cleavage site, 3C protease cleavage site, TEV cleavage site, PreScission protease cleavage site, localizing enzyme cleavage site, MMP-12 cleavage site, MMP-13 cleavage site, MMP-17 cleavage site, MMP-20 cleavage site, granzyme-B cleavage site, enterokinase cleavage site, SUMO protease cleavage site, Factor Xa cleavage site, and TVMV protease cleavage site. In some embodiments, the specific protease cleavage site is selected from: thrombin cleavage site, 3C protease cleavage site, and TEV cleavage site.

[0092] Some non-limiting examples of site-specific exogenous proteases and their cleavage recognition sequences that can be used according to this disclosure include: thrombin, TEV protease, 3C protease, SUMO protease, enterokinase, Factor Xa, TVMV protease, etc. Table 1 provides commonly used cleavage site protein sequences of site-specific proteases (non-limiting examples, SEQ ID Nos: 8-18).

[0093] Table 1: Enzyme name, restriction sequence and cleavage site labeling

[0094] In some embodiments, the amino acid sequence of the thrombin cleavage site includes LVPRGS (SEQ ID NO:8), LVPRGF (SEQ ID NO:9), or MYPRGN (SEQ ID NO:10); the amino acid sequence of the 3C protease cleavage site includes LEVLFQGP (SEQ ID NO:11); the amino acid sequence of the TEV cleavage site includes ENLYFQG (SEQ ID NO:12) or ENLYFQS (SEQ ID NO:13); the amino acid sequence of the enterokinase cleavage site includes DDDDK (SEQ ID NO:14); the amino acid sequence of the SUMO protease cleavage site includes EQIGG (SEQ ID NO:15); the amino acid sequence of the Factor Xa cleavage site includes IEGR (SEQ ID NO:16) or IDGR (SEQ ID NO:17); and the amino acid sequence of the TVMV protease cleavage site includes ETVRFQS (SEQ ID NO:18). In some embodiments, the amino acid sequence of the thrombin cleavage site includes LVPRGS (SEQ ID NO:8). In some embodiments, the amino acid sequence of the TEV cleavage site includes ENLYFQG (SEQ ID NO:12).

[0095] In some embodiments, these specific cleavage sites can be inserted into the linker region between the light and heavy chains of the A2 toxin, defined as the space between two cysteine ​​residues forming the disulfide bond between the light and heavy chains (Cys 430-Cys 454, based on the natural A2 sequence NCBI ID:WP_012703873.1, SEQ ID No: 87). A specific non-limiting example of the natural sequence is: ---CVRGIIPFKTKSLDEGYNKALNDLC---. The full-length structure of the A2 protein has not been reported. The inventors used protein structure prediction tools (such as Google Alpha-Fold 3) to predict that this sequence should be a flexible region without structure. Therefore, the specific protease recognition sequence in Table 1 can be added at any position within this linker region. Furthermore, considering that the cleavage site needs to be exposed on the surface as much as possible, the preferred position is in the middle, such as a site inside the -PFKTKSLDEGYNKA- segment.

[0096] In some embodiments, the modified A2 botulinum toxin contains one or more specific protease cleavage sites between positions 432 and 452, between positions 434 and 450, between positions 436 and 448, between positions 438 and 446, or between positions 440 and 444.

[0097] In some embodiments, the modified A2 botulinum toxin further comprises flanking sequences F1 and optional F2 between cysteine ​​residues 430 and 454, wherein F1 is located on one side of the specific protease cleavage site, and F2, when present, is located on the other side of the specific protease cleavage site, and F1 and F2 independently comprise the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 Among them, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12 They exist or do not exist independently of each other, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12 It is selected independently from glycine, serine, and alanine.

[0098] Additional amino acids can be added to both sides of specific cleavage sites to increase the length of the linker region, making these specific cleavage sites more exposed and improving the efficiency of protease recognition and cleavage. The main consideration for choosing additional amino acids is that their side chains are small, making it less likely for new interactions and secondary structures to form. Using amino acids such as G, S, and A to design protein linkers is a common and widely used biochemical method; researchers can generally design them, and different combinations can be expected to achieve similar effects. For example, some non-limiting examples that can be used according to this disclosure (taking 3C protease, thrombin, and TEV enzyme sites as examples):

[0099] (3C protease) --GAGGAGS-LEVLFQGP-AGGS-- and --GAGGAGS-LEVLFQGP-AGAG--;

[0100] (Thrombin) --GAGGAGSLVPRGSAGGS-- and --GAGGAGSLVPRGSAGAG--;

[0101] (TEV enzyme)--GAGGAGSENLYFQGAGGS--and--GAGGAGSENLYFQGAGAG--.

[0102] In some embodiments, the modified A2 botulinum toxin further comprises flanking sequences F3 and optional F4 between cysteine ​​residues 430 and 454, wherein F3 is located on one side of the specific protease cleavage site, and F4, when present, is located on the other side of the specific protease cleavage site, and F3 and F4 independently comprise the amino acid sequence Y1Y2Y3Y4Y5Y6Y7Y8Y9Y 10 Y 11 Y 12 , among which, Y4, Y5, Y6, Y7, Y8, Y9, Y 10 Y 11 Y 12 They exist or do not exist independently of each other, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y 10 Y 11 Y 12 The amino acids are independently selected from glycine, serine, alanine, and hydrophilic amino acids, wherein the proportion of hydrophilic amino acids in F3 is less than 50%, 40%, 30%, 20%, or 10%, and the proportion of hydrophilic amino acids in F4 is less than 50%, 40%, 30%, 20%, or 10%.

[0103] In some embodiments, the hydrophilic amino acids include lysine, arginine, glutamic acid, glutamine, aspartic acid, asparagine, threonine, tyrosine, histidine, and cysteine; optionally, the hydrophilic amino acids include lysine, arginine, glutamic acid, and aspartic acid.

[0104] Furthermore, the inventors unexpectedly discovered that adding a small amount of hydrophilic amino acids (such as K, R, E, D) to the G, S, A combination can improve the solubility of the A2 toxin protein. This is likely because this unstructured linker region is exposed to the surface and solution, so increasing the hydrophilicity of this sequence can increase protein solubility. For example, some non-limiting examples that can be designed based on this disclosure (taking 3C protease, thrombin, and TEV enzyme sites as examples):

[0105] (3C protease)-AGAGKS-LEVLFQGP-AGAG- and -AGAGKS-LEVLFQGP-AGGS-;

[0106] (thrombin)-AGAGKSLVPRGSAGAG- and -AGAGKSLVPRGSAGGS-;

[0107] (TEV enzyme)-AGAGKSENLYFQGAGAG- and -AGAGKSENLYFQGAGGS-.

[0108] Some non-limiting examples that can be designed based on this disclosure are included in the sequence list SEQ ID Nos: 1-7.

[0109] In some embodiments, F1 and F2 are independently selected from GAGGAGS (SEQ ID NO:1), AGGS (SEQ ID NO:2), AGAG (SEQ ID NO:3), GSAGGS (SEQ ID NO:4), GSAGAG (SEQ ID NO:5), and GAGAG (SEQ ID NO:6).

[0110] In some embodiments, F3 and F4 are independently selected from GAGGAGS (SEQ ID NO:1), AGGS (SEQ ID NO:2), AGAG (SEQ ID NO:3), GSAGGS (SEQ ID NO:4), GSAGAG (SEQ ID NO:5), GAGAG (SEQ ID NO:6), and AGAGKS (SEQ ID NO:7).

[0111] In some implementations, the added sequence may not alter the original native A2 linker sequence; it may simply be an insertion. It may also replace one or more native amino acids. Researchers can generally design insertion sites according to the disclosed principles. For example, some non-limiting examples that can be designed based on this disclosure include: ---CVRGIIPFKTK-insert sequence-SLDEGYNKALNDLC---; ---CVRGIIPFKTK-insert sequence-GYNKALNDLC---; ---CVRGIIPFKTK-insert sequence-ALNDLC---; ---CVRGIIPFK-insert sequence-DEGYNKALNDLC---; ---CVRGIIPFKTK-insert sequence-KALNDLC---; ---CVRGII-insert sequence-KALNDLC---; ---CVRGIIP-insert sequence-KALNDLC---, etc. Examples of insertion sequences can be any of the sequences described above and include those in Table 1.

[0112] In some embodiments, the modified A2 botulinum toxin contains one or more of the following amino acid sequences between cysteine ​​residues at positions 430 and 454:

[0113] (1) KGAGGAGSLVPRGSAGGSA (SEQ ID NO: 19);

[0114] (2) KAGAGKSLVPRGSAGAGA (SEQ ID NO: 20);

[0115] (3) KGAGGAGSLVPRGSAGAGA (SEQ ID NO: 21);

[0116] (4) KAGAGKSLVPRGSAGGSA (SEQ ID NO: 22);

[0117] (5)KGAGGAGSLVPRGFAGGSA(SEQ ID NO:23);

[0118] (6)KAGAGKSLVPRGFAGAGA(SEQ ID NO:24);

[0119] (7)KGAGGAGSLVPRGFAGAGA(SEQ ID NO:25);

[0120] (8)KAGAGKSLVPRGFAGGSA(SEQ ID NO:26);

[0121] (9)KGAGGAGSMYPRGNAGGSA(SEQ ID NO:27);

[0122] (10)KAGAGKSMYPRGNAGAGA(SEQ ID NO:28);

[0123] (11)KGAGGAGSMYPRGNAGAGA(SEQ ID NO:29);

[0124] (12)KAGAGKSMYPRGNAGGSA(SEQ ID NO:30);

[0125] (13)KGAGGAGSLEVLFQGPAGGSA(SEQ ID NO:31);

[0126] (14)KAGAGKSLEVLFQGPAGAGA(SEQ ID NO:32);

[0127] (15)KGAGGAGSLEVLFQGPAGAGA(SEQ ID NO:33);

[0128] (16)KAGAGKSLEVLFQGPAGGSA(SEQ ID NO:34);

[0129] (17)KGAGGAGSENLYFQGAGGSA(SEQ ID NO:35);

[0130] (18)KAGAGKSENLYFQGAGAGA(SEQ ID NO:36);

[0131] (19)KGAGGAGSENLYFQGAGAGA(SEQ ID NO:37);

[0132] (20)KAGAGKSENLYFQGAGGSA(SEQ ID NO:38)。

[0133] In some embodiments, the modified A2 botulinum toxin contains one or more of the following between cysteine ​​residues: (1) KGAGGAGSLVPRGSAGGSA (SEQ ID NO:19), (2) KAGAGKSLVPRGSAGAGA (SEQ ID NO:20), (14) KAGAGKSLEVLFQGPAGAGA (SEQ ID NO:32), and (18) KAGAGKSENLYFQGAGAGA (SEQ ID NO:36). In some embodiments, the modified A2 botulinum toxin further comprises one or more amino acid mutations that do not result in a reduction in the activity of the modified A2 botulinum toxin in inhibiting neuronal activity and / or reducing muscle strength in mammals, said reduction being relative to the amino acid sequences of NCBI ID:WP_012703873.1 (SEQ ID NO:87), NCBI ID:ABC26002 (SEQ ID NO:102), NCBI ID:ADB85243 (SEQ ID NO:98), NCBI ID:ADN04908 (SEQ ID NO:105), NCBI ID:AFM77673.1 (SEQ ID NO:100), NCBI ID:AFV13855.1 (SEQ ID NO:99), NCBI ID:AIT75847.1 (SEQ ID NO:97), and NCBI ID:AJE13255 (SEQ ID NO:99). Regarding the corresponding activities of the amino acid sequences of NCBI ID: KEI94032.1 (SEQ ID NO:101), NCBI ID: KEI94982 (SEQ ID NO:103), NCBI ID: NFD83166.1 (SEQ ID NO:110), NCBI ID: NFJ22948.1 (SEQ ID NO:107), NCBI ID: WP_079007110 (SEQ ID NO:106), NCBI ID: WP_205607627 (SEQ ID NO:109), and / or NCBI ID: WP_228605784 (SEQ ID NO:108), NCBI ID: KEI94032.1 (SEQ ID NO:101), NCBI ID: KEI94982 (SEQ ID NO:103), NCBI ID: NFD83166.1 (SEQ ID NO:110), NCBI ID: NFJ22948.1 (SEQ ID NO:107), NCBI ID: WP_079007110 (SEQ ID NO:106), NCBI ID: WP_205607627 (SEQ ID NO:109), and / or NCBI ID: WP_228605784 (SEQ ID NO:108).

[0134] In some implementations, the recombinant A2 protein sequence may also have common affinity purification tags added to its N-terminus or C-terminus to aid in subsequent protein purification. An "affinity tag" is a polypeptide sequence that can specifically bind to a substance or part of it, such as one containing a peptide bond with Ni. 2+ Affinity tags that specifically bind to multiple histidine residues. These tags can be attached to proteins to facilitate their separation. Affinity tags are typically fused to proteins using recombinant DNA techniques known to those skilled in the art. The use of affinity tags to facilitate protein separation is also well-known in the art. Suitable affinity tags that may be used according to this disclosure include, but are not limited to, His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Soffag 1, Softag 3, TC, V5, VSV, Xpress, Halo, and Fc. In some embodiments, specific protease cleavage sites (e.g., Table 1) may be inserted between the affinity tag and the toxin protein, allowing for the separation of the affinity tag and toxin protein by incubation with a specific protease, thus reducing the potential impact of the affinity tag sequence on toxin activity. For example, some non-limiting examples of sequences that may be used according to this disclosure are listed in the sequence list.

[0135] In some embodiments, the modified A2 botulinum toxin includes an affinity tag at its N-terminus and / or C-terminus, the affinity tag comprising one or more of His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Soffag 1, Softag 3, TC, V5, VSV, Xpress, Halo, and Fc. In some embodiments, the affinity tag includes His6. In some embodiments, the affinity tag comprises an amino acid sequence as shown in SEQ ID NO:96.

[0136] In an optional implementation, the recombinant A2 protein sequence does not contain any additional amino acids at the N-terminus and C-terminus. There are no affinity-purified tag sequences. The advantage of this is that it maintains consistency with both ends of the native A2, thus minimizing any impact on activity. The disadvantage is the relatively low yield of protein purification. Currently, there are no reports in the published literature of recombinant production of A2 protein without any affinity tags. The inventors have successfully expressed and purified this designed protein for the first time and verified that its activity reaches or exceeds that of the native A2 toxin purified from Clostridium botulinum.

[0137] In some embodiments, the modified A2 botulinum toxin does not contain affinity tags at either the N-terminus or the C-terminus. The affinity tags include one or more of His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Soffag 1, Softag 3, TC, V5, VSV, Xpress, Halo, and Fc. In some embodiments, the modified type A2 botulinum toxin is expressed in the amino acid sequences of NCBI ID:WP_012703873.1 (SEQ ID NO:87), NCBI ID:ABC26002 (SEQ ID NO:102), NCBI ID:ADB85243 (SEQ ID NO:98), NCBI ID:ADN04908 (SEQ ID NO:105), NCBI ID:AFM77673.1 (SEQ ID NO:100), NCBI ID:AFV13855.1 (SEQ ID NO:99), NCBI ID:AIT75847.1 (SEQ ID NO:97), NCBI ID:AJE13255 (SEQ ID NO:104), and NCBI ID:KEI94032.1 (SEQ ID NO:101). The amino acid sequences of the following NCBI IDs contain one or more specific protease cleavage sites between cysteine ​​residues: ID: KEI94982 (SEQ ID NO: 103), ID: NFD83166.1 (SEQ ID NO: 110), ID: NFJ22948.1 (SEQ ID NO: 107), ID: WP_079007110 (SEQ ID NO: 106), ID: WP_205607627 (SEQ ID NO: 109), and / or ID: WP_228605784 (SEQ ID NO: 108).

[0138] In some implementations, the optional A2 sequence includes all currently known natural sequences belonging to A2. A total of 15 unique A2 protein sequences have been reported. These sequences are highly similar, with differences between them less than 2.6%. These subtle sequence differences are generally considered to be unavoidable due to the relatively high mutation rate in bacteria. Functionally impactful mutations are generally not retained. These retained natural mutations generally occur at relatively unimportant sites and do not affect various aspects of the protein's properties. For example, the amino acid sequences of the linker region between the light and heavy chains are identical in all A2 sequences, and the cysteine ​​positions are also the same (positions 430 and 454). Therefore, the design and discovery based on a representative A2 sequence has good reason to be generalized to all A2 sequences. Table 2 lists the NCBI search numbers of the 15 natural A2 sequences. Some non-limiting examples of sequences that can be designed based on this disclosure cover these 15 A2 sequences with added specific restriction enzyme sites and corresponding added sequences in the appendix list (SEQ ID Nos: 39-86, 88-95).

[0139] Table 2: Unique NCBI Registry Numbers for Natural A2 Sequences

[0140] In some embodiments, the modified type A2 botulinum toxin comprises an amino acid sequence shown in any of SEQ ID NOs:39-86, 88-95, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity with the amino acid sequence shown in any of SEQ ID NOs:39-86, 88-95.

[0141] Optional A2 sequences further include A2 sequences that have been artificially modified with point mutations, both known and future, based on existing A2 sequences. These point mutations can be in the light chain, linker region, or heavy chain. They can also involve adding amino acids or peptides to the N-terminus or C-terminus of the A2 sequence. The purpose can be to regulate activity, protein stability, protein solubility, isoelectric point, protease sensitivity, etc. Neutral mutations that do not affect function are also included. According to the subtype definition, any sequence differing from the existing natural A2 protein by less than 2.6% is classified as A2, and the contents disclosed in this patent have sufficient grounds for general application to these artificially modified A2 sequences. The methods for comparing protein sequence differences are generally well-known to researchers.

[0142] In some embodiments, the modified A2 botulinum toxin is capable of inhibiting neuronal activity in mammals. In some embodiments, the mammals include humans or rodents, such as rats or mice.

[0143] In some embodiments, the modified A2 botulinum toxin can reduce muscle strength in mammals. In some embodiments, the mammals include humans or rodents, such as rats or mice.

[0144] Currently, there are no publicly reported cases of recombinantly producing A2 protein with the same activity as natural A2 botulinum toxin. Producing A2 protein using recombinant methods is achievable. However, whether the produced A2 protein will reach the level of natural A2 protein purified from Clostridium botulinum is unknown. A key point here is how to activate it with a protease without affecting the protein titer. Natural A2 protein is activated by proteases from Clostridium botulinum under the protection of a complex.

[0145] The method for introducing specific restriction enzyme sites and the specific design of these sites disclosed herein provide the possibility for recombinant production and activation. The recombinant A2 protein expressed and purified using this method exhibits, for the first time, activity in mice that reaches or exceeds that of naturally occurring A2 botulinum toxin extracted from Clostridium botulinum.

[0146] Nucleic acid, vector, host cell

[0147] The second aspect of this disclosure provides a nucleic acid molecule encoding the modified A2 type botulinum toxin of the first aspect of this disclosure.

[0148] A third aspect of this disclosure provides an expression vector comprising the nucleic acid molecule of the second aspect of this disclosure.

[0149] The fourth aspect of this disclosure provides a host cell comprising a nucleic acid molecule of the second aspect of this disclosure or an expression vector of the third aspect of this disclosure.

[0150] In some embodiments, the host cell is selected from Escherichia coli cells, Bacillus subtilis cells, yeast cells, insect cells, and mammalian cells. In some embodiments, the host cell is Escherichia coli.

[0151] Common expression cell methods and systems for recombinant protein production include those using *E. coli*, *Bacillus subtilis*, yeast, insect cells, and mammalian cells (such as CHO cells). These expression systems are commonly used methods and are widely applied in literature and practice for the expression of various proteins. They are all familiar to researchers.

[0152] In some embodiments, *E. coli* is chosen as the expression host for the recombinant expression system. The nucleic acid molecule encoding the modified A2 botulinum toxin protein described in any of the preceding embodiments can be produced through gene synthesis. Simultaneously, the gene encoding is optimized for *E. coli* to increase expression levels. Gene synthesis and encoding optimization are common methods familiar to researchers. The synthesized toxin gene is then placed into a suitable expression vector. Several common expression vectors can be used with *E. coli* as the expression host. In optional embodiments, the expression vector has a promoter selected from Tac, AraBAD, T7-Lac, or T5-Lac. The expression vector may include (but is not limited to) common *E. coli* expression vectors such as pET, pGEX, and pJ401.

[0153] Preparation method

[0154] The fifth aspect of this disclosure provides a method for preparing modified A2 botulinum toxin, comprising culturing host cells of the fourth aspect of this disclosure under conditions favorable to the expression of the modified A2 botulinum toxin of the first aspect of this disclosure.

[0155] Methods and techniques for lysing host cells, such as bacterial cells, especially *E. coli* host cells, are known in the art. Examples include sonication or the use of a Freund's crusher. Purification of toxins may involve one or more precipitation and extraction steps, one or more concentration steps, and other various chromatographic steps.

[0156] The sixth aspect of this disclosure provides a method for producing soluble double-stranded type A2 botulinum toxin, the method comprising:

[0157] Step (1): Contact the modified A2 botulinum toxin of the first aspect of this disclosure or the modified A2 botulinum toxin prepared by the preparation method of the fifth aspect of this disclosure with a specific protease capable of enzymatically cleaving the specific protease cleavage site.

[0158] In some embodiments, the method further includes:

[0159] Step (2): Contact the product of step (1) with a hydrophobic surface to separate and obtain the soluble double-stranded type A2 botulinum toxin.

[0160] In some embodiments, the hydrophobic surface comprises an inert matrix with ligands composed of aryl or alkyl groups attached thereto. In some embodiments, the ligands are selected from butyl, phenyl, or octyl ligands.

[0161] In some embodiments, the method includes incubating the crudely purified recombinantly expressed modified A2 toxin protein with a corresponding protease for cleavage, based on the introduced specific protease cleavage site. The protease may be added at multiple steps in the purification process from cell lysates. For example, it may be added before or after protein precipitation, ion exchange chromatography, hydrophobic interaction chromatography, and / or size exclusion chromatography. The contact step requires incubation at conditions and time sufficient for the protease to cleave the A2 protein. In one aspect, the "sufficient cleavage time" depends on the degree of cleavage that the proteolytically processed polypeptide or composition containing it should have. In one aspect, the method includes incubating the protease and A2 protein for at least 30 minutes, 60 minutes, 120 minutes, or at least 240 minutes. In another aspect, the protease and A2 protein are incubated for up to 30 minutes, 60 minutes, 120 minutes, 240 minutes, 480 minutes, or up to 600 minutes. In yet another aspect, the method includes incubating the protease and A2 protein at 4°C or 37°C. In another aspect, the method includes steps of incubation for up to 1 hour, up to 2 hours, 4 hours, 6 hours, 10 hours, or up to 16 hours.

[0162] The double-stranded A2 botulinum toxin protein is then isolated by contacting a solution containing type A2 botulinum toxin protein and a protease with a hydrophobic surface. In an optional embodiment, the hydrophobic surface comprises an inert matrix with ligands composed of aryl or alkyl groups attached thereto. In some embodiments, the ligands are selected from butyl, phenyl, or octyl ligands.

[0163] Composition

[0164] The seventh aspect of this disclosure provides a composition comprising soluble double-stranded type A2 botulinum toxin prepared using the preparation method of the sixth aspect of this disclosure.

[0165] This disclosure also provides a pharmaceutical composition comprising, as an active ingredient, a double-stranded type A2 botulinum toxin protein prepared using the preparation method described in the sixth aspect of this disclosure.

[0166] In some embodiments, the composition is a liquid or lyophilized powder composition.

[0167] The composition typically comprises a mixture of activated double-stranded A2 and residual unactivated single-stranded A2. In some embodiments, the composition contains less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of single-stranded A2 botulinum toxin, based on the total weight of the composition. In a preferred embodiment, the mixture contains less than 5% of unactivated single-stranded A2, such as less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0168] Uses and methods

[0169] The eighth aspect of this disclosure provides the use of the modified A2 botulinum toxin of the first aspect of this disclosure, the modified A2 botulinum toxin prepared by the preparation method of the fifth aspect of this disclosure, the soluble double-stranded A2 botulinum toxin prepared by the preparation method of the sixth aspect of this disclosure, or the composition of the seventh aspect of this disclosure in the preparation of cosmetic products.

[0170] The ninth aspect of this disclosure provides the use of the modified A2 botulinum toxin of the first aspect of this disclosure, the modified A2 botulinum toxin prepared by the method of the fifth aspect of this disclosure, the soluble double-stranded A2 botulinum toxin prepared by the method of the sixth aspect of this disclosure, or the composition of the seventh aspect of this disclosure in the preparation of a drug for reducing neuronal activity.

[0171] In some embodiments, the drug is used to treat upper limb spasticity, lower limb spasticity, post-stroke spasticity, spastic dysphonia, spasmodic torticollis, laryngeal dystonia, orofacial dystonia, tongue dystonia, neck dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spastic colitis, neurogenic bladder, anal spasticity, limb spasticity, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, pain caused by muscle spasms, skin conditions, tremor, migraine, psoriasis, allergic reactions, erythrocytic lymphohistiocytosis, and alcoholic pancreatic disease.

[0172] In a tenth aspect, this disclosure provides a method for treating, preventing, or alleviating a disease, symptom, or disorder, the method comprising: administering to a subject in need a therapeutically effective amount of a modified A2 botulinum toxin of the first aspect of this disclosure, a modified A2 botulinum toxin prepared using the preparation method of the fifth aspect of this disclosure, a soluble double-stranded A2 botulinum toxin prepared using the preparation method of the sixth aspect of this disclosure, or a composition of the seventh aspect of this disclosure.

[0173] In some embodiments, the disease, symptom, or disorder includes one or more of the following: upper limb spasticity, lower limb spasticity, post-stroke spasticity, spontaneous spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, orofacial dysphonia, tongue dystonia, neck dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasm and other voice disorders, spastic colitis, neurogenic bladder, anal spasm, limb spasticity, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, pain caused by muscle spasm, dermatological conditions, tremor, migraine, psoriasis, allergic reactions, erythrocytic lymphohistiocytosis, alcoholic pancreatic disease, and glabellar lines.

[0174] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. The descriptions of the exemplary embodiments are merely illustrative and are not intended to limit the scope of the disclosure or its application or use. The present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to make the present disclosure thorough and complete, and to fully express the scope of the disclosure to those skilled in the art. It should be noted that, unless specifically stated otherwise, the technical means described in these embodiments should be interpreted as exemplary only and not restrictive. The following embodiments illustratively illustrate the technical solutions of the present disclosure but do not constitute any limitation on the scope of protection of the present disclosure.

[0175] Example 1: Construction and transformation of modified A2 botulinum toxin plasmids containing thrombin sites, 3C protease sites, or TEV enzyme sites.

[0176] To explore whether the A2 toxin protein could be produced using recombinant methods and achieve the activity of the natural toxin, the inventors designed a method to add a specific protease recognition site sequence to the linker region between the light and heavy chains of the A2 protein. In this embodiment, thrombin, the 3C protease site, or the TEV protease site were selected as the specific protease. Additional sequences were also added flanking most of the restriction enzyme sites. Based on the amino acid sequences published in the sequence list, the DNA sequence was synthesized and then inserted into an *E. coli* expression vector (pET28a(+)) to obtain the recombinant plasmid. The design, synthesis, and cloning of the DNA coding sequence into the *E. coli* expression vector are all routine experimental procedures that can be successfully performed by those skilled in the art.

[0177] The transformation of E. coli was carried out according to the following steps: 2 μL of the synthesized expression plasmid was added to competent E. coli expression strain (BL21(DE3), commercially available), incubated on ice for 30 min, and then heat-shocked at 42℃ for 90 s; 800 μL of fresh LB medium was added and incubated for 60 min, and then spread on LB solid plates containing kanamycin resistance (components per liter: NaCl 10 g, tryptone 10 g, yeast extract 5 g, agar 10 g, containing 50 μg / mL kanamycin), and cultured overnight at 37℃.

[0178] Induction of expression was performed as follows: Single colonies were picked and inoculated into 10 mL of LB medium (components per liter: NaCl 10 g, tryptone 10 g, yeast extract 5 g, containing 50 μg / mL kanamycin), and cultured overnight at 37°C and 220 rpm. Then, 100 mL of the resulting culture was transferred to 1 L of TB medium (components per liter: soybean peptone 12 g; yeast extract 24 g; Na₂HPO₄ 9.4 g; KH₂PO₄ 2.2 g; glycerol 5 g; pH 7.0-7.2, containing 50 μg / mL kanamycin), and cultured at 37°C and 220 rpm until OD₂O₃ was reached. 600 Between 0.6 and 0.8, the temperature was lowered to 20°C, and IPTG at a final concentration of 1 mM was added to induce expression for 20 hours.

[0179] The amino acid sequence of the modified A2 type botulinum toxin and its sequence design description are shown in Table 3.

[0180] Table 3: Amino acid sequences and their sequence design descriptions

[0181] Example 2: Purification and enzymatic activation of recombinant A2 toxin protein containing thrombin sites

[0182] The fermented bacterial broth was centrifuged at 4800 rpm and 4°C for 20 min to collect wet bacterial cells. The collected bacterial cells were resuspended at a ratio of 10% and then lysed using a high-pressure homogenizer. The lysed bacterial cells underwent a clarification step and were then separated and removed from other cellular components by ammonium sulfate precipitation. Then, the botulinum toxin protein was purified and separated using a Fast protein liquid chromatography (FPLC) system (refer to Tepp WH, Lin G, Johnson EA. Purification and characterization of a novel subtype a3 botulinum neurotoxin. Appl Environ Microbiol. 2012 May; 78(9):3108-13. doi:10.1128 / AEM.07967-11. Epub 2012 Feb 24. PMID:22367089; PMCID:PMC3346436). The preliminarily purified botulinum toxin protein was incubated with thrombin. The enzyme digestion procedure is as follows: Thrombin stock solution is mixed with the sample to achieve a mass ratio of 1:60 (enzyme:sample), thoroughly mixed, and incubated at 18–26°C for 3 hours to complete enzyme digestion and activation. Thrombin is then removed again by chromatography. Finally, UFDF ultrafiltration is performed to concentrate the sample and replace it with storage buffer to obtain a high-purity botulinum toxin protein solution.

[0183] The purity of the two batches of purified protein samples was analyzed by protein gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining. As shown in Figure 3, sample (A2-4, SEQ ID No:39) was located at 150 kDa under non-reducing conditions, proving that the full-length toxin protein had been purified. Under reducing conditions, the original 150 kDa toxin molecule was separated into two bands of 100 kDa and 50 kDa, indicating that the disulfide bonds connecting the light and heavy chains were broken. The reduced sample still had a faint 150 kDa band in the lane, indicating that the sample was not fully digested, accounting for less than 2% of the total sample, indicating that the enzyme digestion activation degree of the sample was as high as 98%. The purity of the protein was further analyzed using the commonly used size exclusion chromatography (SEC-HPLC) method (Figure 4). The sample showed a main peak in the SEC-HPLC detection, and the purities of the two batches of samples were 99.4% and 99.1%, respectively (Table 4).

[0184] Table 4: Purity analysis results of two batches of purified protein samples using SDS-PAGE and SEC-HPLC methods, respectively.

[0185] Example 3: Measurement of the bioactivity (LD50) of purified recombinant A2 botulinum toxin containing thrombin sites 50 experiment)

[0186] The biological activity (toxicity) of botulinum toxin is crucial for its use in clinical trials. Currently, the internationally accepted detection method remains determining the median lethal dose (LD50) of the toxin in mice. 50 This experiment used 280 female CD-1 mice, ensuring their weight was between 20 and 24 g. The animals underwent an acclimatization period of 3–7 days upon arrival at the facility. During this period, their health was monitored daily. Any mice exhibiting abnormalities or infections were removed from the experimental group. Before the experiment, the 280 mice were weighed and randomly assigned to groups. On the day of the experiment, the toxin concentrate was diluted. The diluent consisted of ordinary physiological saline plus 0.5% human serum albumin. Each group of mice was then intraperitoneally injected with 0.5 ml of the test substance at different doses listed in the table below (A2-4). Animal mortality was observed and recorded within 96 hours post-injection, and the median lethal dose (theoretically the dose that causes 50% mortality in mice) was calculated. LD 50 Numerical values ​​are generally expressed in units per milligram of toxin protein (U / mg). One unit (U) is defined as 1 LD. 50 This is the median lethal dose for mice.

[0187] Table 5: LD 50 Experimental results

[0188] As shown in Table 5, the two batches of experimental samples (A2-4, SEQ ID No: 39) LD 50 The test results were similar. The first test result was LD. 50 =6.905 pg, confidence interval 6.128–7.938; second test result was LD 50 = 8.310 pg, confidence interval 7.287-9.829. Converted to SI units twice, the result is: 1.44 × 10⁻⁶. 8 U / mg and 1.20×10 8 U / mg. This activity result reaches the activity of natural botulinum toxin type A extracted from Clostridium botulinum. For example, the literature (Kohda et al, Microbiology and Immunology, 2020, 64:502-511) reports the activity of natural botulinum toxin type A in mice with LD50. 50 The method detection result for type A1 is 8.8 × 10⁻⁶. 7 U / mg, the natural A2 type is 1.2×10 8U / mg. This paper also reports that the activity of the A2 type toxin produced by recombinant E. coli is 1.25 × 10 U / mg. 7 U / mg, which is 10 times less active than the natural toxin. In contrast, our designed and recombinant purified A2 botulinum toxin has, for the first time, achieved activity comparable to that of natural A2 botulinum toxin extracted from Clostridium botulinum.

[0189] Example 4: Comparison of the efficacy of recombinant A2 type and natural A1 type toxins using mouse behavioral hind toe abduction score (DAS) experiment.

[0190] The in vivo efficacy of neurotoxins can be determined by the hind toe abduction score (DAS) (a measure of muscle paralysis). Before injection of the test substance, a DAS test was performed, and the animals were anesthetized with isoflurane gas. Then, the corresponding test substance was injected into the mid-section of one gastrocnemius muscle of each group of mice, while a blank control reagent was injected into the mid-section of the other gastrocnemius muscle. DAS tests were performed at 0h, 4h, 8h, and 24h post-administration, and mouse weight was recorded every 24 hours thereafter, until the DAS score disappeared.

[0191] DAS experimental protocol: To explore whether recombinant A2 toxin containing thrombin sites has an efficacy difference compared to commonly used A1 toxin, the inventors selected the most representative product with natural A1 as the active ingredient, brand name Botox. As a parallel comparison of recombinant purified A2 activated with thrombin, the inhibitory effect of the test product on defensive hind limb finger abduction in mice was observed by injecting Botox and recombinant A2 protein (Table 5). During the procedure, the mouse tail was suspended and rapidly lowered to induce a physiological conditioned reflex, causing the five fingers of the hind limbs to separate. After injection of Botox and recombinant A2 toxin, the DAS response was assessed by two separate observers using a double-blind, five-point scale. The DAS score ranged from 0 to 4: 0 for normal, completely separated fingers; 1 for fingers divided into four groups (two fingers fused together); 2 for fingers divided into three groups (two fingers fused together, or one finger fused into three); 3 for fingers divided into two groups (one fused into two and one fused into three, or one fused into four); and 4 for all fingers fused together and unable to open. The operator observed and recorded the DAS response score for each dose. Each experiment needs to be repeated 6 times to obtain the average value.

[0192] Table 6: Grouping and Dosage Design in DAS Behavioral Experiments

[0193] The hind toe abduction score (DAS) of female CD-1 mice was observed and recorded by intramuscular injection of the sample (A2-4, SEQ ID No: 39) and its effect on mouse body weight: the decrease in mouse body weight reflected the diffusion of botulinum toxin from the injection site to the whole body. The DAS test results are shown in Figure 5. The results showed that DAS scores were generated 4 hours after injection of recombinant A2 and Botox. The DAS scores increased over time until they began to decline slowly 2 or 3 days after administration, and in a dose-dependent manner. By day 18 after administration, the DAS response of the mice had basically returned to normal levels. The results of comparing A1 and A2 botulinum toxins at different doses showed that: (1) the onset time of recombinant A2 botulinum toxin was faster than that of natural A1; (2) the efficacy of recombinant A2 botulinum toxin was higher than that of A1: the DAS score of A2 was higher than that of A1 at all three doses; (3) the systemic toxicity of recombinant A2 botulinum toxin was less than that of A1: the weight loss caused by recombinant A2 toxin was less than that caused by A1 at all three doses (Figure 6), indicating that recombinant A2 toxin had less systemic diffusion toxicity and was safer.

[0194] Example 5: Electrophysiological measurement of mouse muscle strength to compare the efficacy of recombinant A2 type and natural A1 type toxins.

[0195] Electrophysiological measurements of mouse leg muscles directly can more accurately reflect the muscle paralysis effect of botulinum toxin in vivo. Before the experiment, mice were weighed and randomly grouped. Animals were anesthetized with isoflurane gas before drug administration, followed by an injection of 5 μL of the corresponding test substance into the mid-section of the left hind leg gastrocnemius muscle of each group of mice. Animal survival status and body weight were monitored daily. The day of drug administration was defined as D0. Muscle strength was tested in mice on days D1, D3, D7, D14, and D21 using a 1300A muscle strength recorder. First, each animal was anesthetized by intraperitoneal injection of 10% chloral hydrate. The hair on the leg muscles of the mice was then removed. The mice were then fixed in a supine position on the test plate with their paws in contact with the sensor pedals of the test system and secured with tape. The hind limbs of the mice were then fixed to the paws at an angle of 90 degrees, and alcohol was applied to the gastrocnemius muscle for disinfection. Two stimulation electrodes were then inserted subcutaneously into the gastrocnemius muscle to avoid injuring tendons and ligaments. The gastrocnemius muscle group was electrically stimulated at a stimulation interval of 90 seconds, a stimulation frequency of 150 Hz, a voltage intensity of 5 V, and a stimulation duration of 900 ms. The muscle strength of the mouse's gastrocnemius muscle was obtained and subsequent experimental analysis was performed.

[0196] The muscle strength test results for sample (A2-4, SEQ ID No: 39) are shown in Figure 7. Female CD-1 mice were injected into the gastrocnemius muscle, and muscle strength was measured at different time points. The results showed that injection of recombinant A2 toxin at three different doses achieved better results than injection of A1 type (Botox). The greater decrease in muscle strength further proves that the in vivo efficacy of recombinant A2 toxin is higher than that of A1 toxin.

[0197] Example 6: Sequence Screening of Recombinant Type A2 Botulinum Toxin

[0198] Example 6.1: Protein purification and enzymatic activation

[0199] Among the currently discovered natural sequences of type A2 botulinum toxin, three sequences were selected for recombinant design and production: A2-1 (SEQ ID NO: 86), A2-2 (SEQ ID NO: 89), and A2-3 (SEQ ID NO: 90). For the three sequences in this embodiment, affinity chromatography was used for purification. Ni-NTA media from GenScript was used for affinity chromatography. After loading the sample, impurities were removed using 50 mM Tris pH 7.5, 150 mM NaCl, and 10 mM Imidazole solution. Then, elution was performed using 50 mM Tris pH 7.5, 500 mM NaCl, and 60 mM Imidazole solution to obtain the target protein. The affinity-eluted sample was activated by thrombin digestion. SDS-PAGE was used to analyze the protein samples activated after thrombin digestion, distinguishing between reduced and non-reduced samples. Non-reduced samples were loaded using 4×LDS Sample Buffer (GenScript, catalog number: M00676), while reduced samples were mixed with 5×Sample Buffer (GenScript, catalog number: MB01015), heated at 70°C for 5 minutes, and then loaded. The Sample Buffer for reduced samples contained β-mercaptoethanol as a reducing agent, which could break the disulfide bonds between molecules under heating conditions.

[0200] The SDS-PAGE results are shown in Figure 8. After affinity chromatography purification, the non-reduced purities of A2-1 and A2-2 molecules were 93% and 96%, respectively, both exceeding 90%. The purity of the non-reduced sample after purification of A2-3 molecule was 78%. Before enzyme digestion, A2-1, A2-2, and A2-3 all showed a 150 kDa band. After enzyme digestion, the non-reduced sample still showed a 150 kDa band. However, under the reducing conditions after enzyme digestion, the 150 kDa band split into a 100 kDa heavy chain toxin band and a 50 kDa light chain toxin band. This indicates that the above recombinant A2 type botulinum toxins all transformed into a 100 kDa heavy chain and a 50 kDa light chain linked by disulfide bonds after enzyme digestion. This result shows that there is no difference in enzyme digestion activation among the three recombinant A2 type botulinum toxins. In summary, the recombinant A2 type botulinum toxins of this application can all be purified and can be well activated after insertion into the protease cleavage site.

[0201] Example 6.2: Mouse behavioral hind toe abduction score (DAS) experiment

[0202] The potency and duration of activation of A2-1, A2-2, and A2-3 after enzyme digestion were determined by mouse hind toe abduction score (DAS), using the same experimental method as in Example 4. Figure 9 shows the changes in DAS scores of activated A2-1, A2-2, and A2-3 molecules at different doses (18 pg, 9 pg, 4.5 pg, and 2 pg) with injection time.

[0203] As shown in Figure 9(A), the highest dose of A2-1 molecule was 18 pg. All mice died on the third day after administration and could not be observed further. The 9 pg dose group reached the highest score of 3.2 points on the first day; the 4.5 pg dose group reached the highest score of 2.6 points on the second day; and the 2 pg dose group reached the highest score of 2.4 points on the first day.

[0204] As shown in Figure 9(B), the 18 pg dose group of A2-2 molecule reached its highest score of 3.8 on the second day after administration, and the score then decreased over time. The 9 pg dose group reached its highest score of 3.2 on the second day; the 4.5 pg dose group reached its highest score of 2.6 on the first day; and the 2 pg dose group reached its highest score of 2.2 on the second day.

[0205] As shown in Figure 9(C), the 18 pg dose group of molecule A2-3 reached its highest score of 2.8 on the second day after administration, and then the downward trend was similar to that of molecule A2-2. The 9 pg dose group reached its highest score of 2.8 on the second day; the 4.5 pg dose group reached its highest score of 2.2 on the first day; and the 2 pg dose group reached its highest score of 1.8 on the first day.

[0206] In summary, the three molecules in the 9pg dose group showed a consistent decreasing trend, all falling below 1 point by day 12 post-administration. In the 4.5pg dose group, molecules A2-1 and A2-2 reached their highest score of 2.5 points on day 2, while molecule A2-3 reached its highest score of 2.1 points on day 2. In the 2pg dose group, molecules A2-1 and A2-2 reached their highest score of 2.1 points on day 2, while molecule A2-3 reached its highest score of 1.9 points. In the 9pg, 4.5pg, and 2pg dose groups, the DAS scores of the three enzyme-activated toxin molecules showed a consistent decreasing trend over time. In the highest dose group, molecule A2-1 exhibited the strongest toxicity. In the other dose groups, molecules A2-1 and A2-2 showed similar toxicity. Molecule A2-3 showed better toxicity than molecules A2-1 and A2-2 in the 9pg dose group, but its toxicity was weaker than the other two molecules in the 2pg and 4.5pg dose groups.

[0207] Example 7: Screening of Enzyme Removal Sites for Recombinant A2 Botulinum Toxin

[0208] Example 7.1: Protein purification and enzymatic activation

[0209] To explore the effects of inserting different types of restriction enzyme sites on the activity and duration of recombinant A2 botulinum toxin, this embodiment selected three restriction enzyme sites to be designed and inserted into recombinant A2 botulinum toxin. These three sequences contain thrombin restriction sites, 3C protease restriction sites, and TEV restriction sites, respectively. Correspondingly, the three recombinant A2 botulinum toxins were named A2-1 (SEQ ID NO: 86), A2-3C (SEQ ID NO: 92), and A2-TEV (SEQ ID NO: 93). In addition, natural A2 botulinum toxin protein was also recombinantly produced and digested using conventional trypsin and thrombin restriction methods. For the four sequences in this embodiment, affinity chromatography was used for purification (refer to Example 6.1), and the purified and eluted samples were activated by corresponding restriction enzyme digestion treatments. SDS-PAGE was used to analyze the activated protein samples after enzyme digestion, distinguishing between reduced and non-reduced samples. Non-reduced samples were loaded using 4×LDS Sample Buffer (GenScript, catalog number: M00676), while reduced samples were mixed with 5×Sample Buffer (GenScript, catalog number: MB01015), heated at 70°C for 5 minutes, and then loaded. The Sample Buffer for reduced samples contained β-mercaptoethanol as a reducing agent, which could break the disulfide bonds between molecules under heating conditions.

[0210] The SDS-PAGE results are shown in Figure 10. The 150kDa band in the A2-1 reduced sample, A2-3C reduced sample, and A2-natural trypsin reduced sample all essentially disappeared. This indicates that treatment with thrombin in the A2-1 sample, with 3C protease in the A2-3C sample, and with trypsin in the A2 natural sample can cleave and activate over 90% of the toxin protein, transforming it into a 50kDa light chain and a 100kDa heavy chain linked by a single disulfide bond. However, comparing the A2-TEV enzyme-reduced and non-reduced samples, the 150kDa band in the reduced sample only decreased by 50%, indicating that even with the addition of TEV cleavage sites in the recombinant A2 botulinum toxin, TEV cleavage does not achieve a 90% activation efficiency. This demonstrates that the activation efficiency of recombinant A2 botulinum toxin varies depending on the cleavage sites introduced. The comparison between the reduced and non-reduced samples treated with A2-natural thrombin shows that thrombin cannot cleave botulinum toxin without added cleavage sites. When natural botulinum toxin is activated using trypsin, an impurity band appears in the reduced sample compared to the non-reduced sample. This is because the trypsin cleavage sites are too numerous on the natural toxin sequence, causing cleavage outside the linker sequence, making trypsin cleavage more difficult to control in industrial production.

[0211] In summary, the recombinant A2 botulinum toxin of this application can be activated to varying degrees after insertion into the thrombin cleavage site, the 3C protease cleavage site, and the TEV cleavage site. No impurities are generated during activation, and purity can be controlled during industrial production, ensuring safety. In particular, insertion into the thrombin cleavage site and the 3C protease cleavage site results in highly efficient (over 90%) activation.

[0212] Example 7.2: Mouse Behavioral Hind Toe Abduction Score (DAS) Experiment

[0213] The potency and duration of the three activated toxin molecules after enzymatic digestion were determined by mouse hind toe abduction score (DAS), using the same experimental method as in Example 4. Figure 11 shows the changes in DAS scores of activated A2-1, A-3C, and A-TEV molecules at different doses (18 pg, 9 pg, 4.5 pg, and 2 pg) with injection time.

[0214] In the 18 pg dose group, all mice injected with A2-1 died on day 3 post-administration. Mice injected with A2-3C reached their highest score of 4 on day 2, while mice injected with A2-TEV reached their highest score of 3.2 on day 2. In the 9 pg dose group, A2-3C reached its highest score of 3.6 on day 2, higher than the 3.2 in the A2-1 group and the 2.2 in the A2-TEV group. In this dose group, two days after botulinum toxin injection, the DAS scores of mice in all molecule groups decreased over time, and the DAS scores of mice injected with A2-3C were higher than those of the other two molecules on each day. In the lower dose group of 4.5 pg, A2-1 reached a maximum score of 2.6, and A2-3 reached 2.4 on day 2, after which the scores gradually decreased. In the lowest dose group (2 pg), the highest scores achieved by the A2-1 and A2-3C molecules were 2.2 and 2.4, respectively, with a consistent downward trend thereafter. Across all dose groups, mice injected with the A2-TEV molecule had the lowest DAS scores among the three molecules, and the scores in the 2 pg dose group all returned to zero on day six after administration. These results indicate that the activity intensity of recombinant A2 botulinum toxin with different enzyme cleavage sites varies.

[0215] Example 8: Screening of paralinked sequences at recombinant A2 botulinum toxin restriction enzyme sites

[0216] Example 8.1: Protein purification and enzymatic activation

[0217] Generally, the activity and duration of recombinant botulinum toxin are influenced by three main functional regions: the enzyme activity region, the transmembrane region, and the receptor binding region. The sequence of the linker adjacent to the restriction enzyme cleavage site is not expected to affect the activity and duration of recombinant botulinum toxin. However, it may affect the overall activity of recombinant botulinum toxin by influencing protein stability and protein folding conformation. Therefore, to explore the effect of the linker sequence adjacent to the restriction enzyme cleavage site on recombinant botulinum toxin, based on previous experimental selections, this embodiment designed three molecules containing thrombin cleavage sites but with different linker sequences adjacent to the cleavage sites: A2-1 (SEQ ID NO: 86), A2-P2 (SEQ ID NO: 94), and A2-L2 (SEQ ID NO: 95). For the three sequences in this embodiment, A2-1, A2-P2, and A2-L2 were purified using affinity chromatography (refer to Example 6.1). The purified and eluted samples were activated by thrombin cleavage. SDS-PAGE was used to analyze the protein samples activated after thrombin digestion, distinguishing between reduced and non-reduced samples. Non-reduced samples were loaded using 4×LDS Sample Buffer (GenScript, catalog number: M00676), while reduced samples were mixed with 5×Sample Buffer (GenScript, catalog number: MB01015), heated at 70°C for 5 minutes, and then loaded. The Sample Buffer for reduced samples contained β-mercaptoethanol as a reducing agent, which could break the disulfide bonds between molecules under heating conditions.

[0218] The SDS-PAGE results are shown in Figure 12. After affinity chromatography purification, the purity of the A2-1 and A2-L2 unreduced samples was similar, both exceeding 90%, while the purity of the A2-P2 unreduced sample was 75%. Before enzyme digestion, all toxin molecules appeared as a single 150 kDa band. After unreduced digestion, the samples still showed a 150 kDa band. Under reducing conditions after enzyme digestion, the 150 kDa band split into a 100 kDa heavy chain toxin band and a 50 kDa light chain toxin band. This indicates that the three recombinant A2 botulinum toxins containing different linkage sequences all transformed into a 100 kDa heavy chain and a 50 kDa light chain linked by disulfide bonds after enzyme digestion, with consistent transformation and activation efficiencies.

[0219] Example 8.2: Mouse Behavioral Hind Toe Abduction Score (DAS) Experiment

[0220] The potency and duration of the three enzyme-activated toxin molecules were determined by mouse hind toe abduction score (DAS), using the same experimental method as in Example 4. Figure 13 shows the changes in DAS scores of different doses (18 pg, 9 pg, 4.5 pg and 2 or 2.25 pg) of A2-1, A2-P2, and A2-L2 molecules with injection time.

[0221] As shown in Figure 13(A), the highest dose of A2-1 molecule was 18 pg. All mice died on the third day after administration and could not be observed further, with a mortality rate of 100%. The 9 pg dose group reached the highest score of 3.2 points on the second day; the 4.5 pg dose group reached the highest score of 2.6 points on the second day; and the 2 pg dose group reached the highest score of 2.2 points on the second day.

[0222] The 18 pg dose group of the A2-P2 molecule reached its highest score of 4 on the first day after administration, but two mice died on the third day after administration. Subsequent changes in mice were not included in the DAS score reference. As shown in Figure 13(B), the 9 pg dose group reached a score of 4 on the second day after administration, and the score dropped rapidly from the third to the seventh day after administration; the 4.5 pg dose group reached its highest score of 2 on the second day; and the 2.25 pg dose group reached its highest score of 1.8 on the first day.

[0223] As shown in Figure 13(C), the 18 pg dose group of A2-L2 molecules reached its highest score of 4 points on the third day after administration, and the score began to decline on the fourth day. The 9 pg dose group reached its highest score of 3.2 points on the second day; the 4.5 pg dose group reached its highest score of 2.4 points on the first day; and the 2 pg dose group reached its highest score of 2.4 points on the second day.

[0224] On day 7 post-injection, mice in the 9 pg dose group had an average DAS score of 1.2 for all three molecules. In the 4.5 pg and lowest dose groups, the A2-P2 score was slightly lower than the other two molecules. In the 4.5 pg dose group, mice subsequently injected with all three molecules showed a consistent downward trend in DAS scores, all reaching an average score of 1 on day 11 post-injection. In the lowest dose group, A2-1 reached zero on day 16, A2-L2 reached zero on day 14, and the decline in A2-P2 was slower than the other two molecules, reaching zero on day 21 post-injection.

[0225] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure.

Claims

1. Modified type A2 botulinum toxin, wherein, The modified A2 type botulinum toxin contains L C Domain and H N Structural domain, the L C Structural domain and the H N The artificial arrangement between structural domains makes the L C Structural domain and the H N A structural unit whose structural domain can be controlled to fracture under predetermined conditions. Optionally, the L C The last cysteine ​​residue at the C-terminus of the domain and the H N The L domain is artificially positioned between the first cysteine ​​residue at the N-terminus. C Structural domain and the H N A structural unit whose structural domain can be controlled to fracture under predetermined conditions. Optionally, the L C The last cysteine ​​residue at the C-terminus of the domain and the H N The first cysteine ​​residue at the N-terminus of the domain contains one or more specific protease cleavage sites. Optionally, the modified A2 botulinum toxin contains one or more specific protease cleavage sites between cysteine ​​residues 430 and 454, wherein the site is defined by the amino acid sequences of NCBI ID:WP_012703873.1 (SEQ ID NO:87), NCBI ID:ABC26002 (SEQ ID NO:102), NCBI ID:ADB85243 (SEQ ID NO:98), NCBI ID:ADN04908 (SEQ ID NO:105), NCBI ID:AFM77673.1 (SEQ ID NO:100), NCBI ID:AFV13855.1 (SEQ ID NO:99), NCBI ID:AIT75847.1 (SEQ ID NO:97), and NCBI ID:AJE13255 (SEQ ID NO:104). The amino acid sequences of NCBI ID: KEI94032.1 (SEQ ID NO: 101), NCBI ID: KEI94982 (SEQ ID NO: 103), NCBI ID: NFD83166.1 (SEQ ID NO: 110), NCBI ID: NFJ22948.1 (SEQ ID NO: 107), NCBI ID: WP_079007110 (SEQ ID NO: 106), NCBI ID: WP_205607627 (SEQ ID NO: 109), and / or NCBI ID: WP_228605784 (SEQ ID NO: 108) are used as references.

2. The modified A2 type botulinum toxin according to claim 1, wherein, The modified A2 botulinum toxin further includes flanking sequences F1 and optional F2 between cysteine ​​residues 430 and 454, wherein F1 is located on one side of the specific protease cleavage site, and F2, when present, is located on the other side of the specific protease cleavage site, and F1 and F2 independently comprise the amino acid sequence X1X2X3X4X5X6X7X8X9X. 10 X 11 X 12 Among them, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12 They exist or do not exist independently of each other, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12 It is selected independently from glycine, serine, and alanine.

3. The modified type A2 botulinum toxin according to claim 1 or 2, wherein, The modified A2 botulinum toxin further includes flanking sequences F3 and optional F4 between cysteine ​​residues 430 and 454, wherein F3 is located on one side of the specific protease cleavage site, and F4, when present, is located on the other side of the specific protease cleavage site, and F3 and F4 independently comprise the amino acid sequence Y1Y2Y3Y4Y5Y6Y7Y8Y9Y 10 Y 11 Y 12 , among them, Y4, Y5, Y6, Y7, Y8, Y9, Y 10 Y 11 Y 12 They exist or do not exist independently of each other, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y 10 Y 11 Y 12 The amino acids are independently selected from glycine, serine, alanine, and hydrophilic amino acids, wherein the proportion of hydrophilic amino acids in F3 is less than 50%, 40%, 30%, 20%, or 10%, and the proportion of hydrophilic amino acids in F4 is less than 50%, 40%, 30%, 20%, or 10%.

4. The modified A2 type botulinum toxin according to claim 3, wherein, The hydrophilic amino acids include lysine, arginine, glutamic acid, glutamine, aspartic acid, asparagine, threonine, tyrosine, histidine, and cysteine; optionally, the hydrophilic amino acids include lysine, arginine, glutamic acid, and aspartic acid.

5. The modified type A2 botulinum toxin according to any one of claims 1 to 4, wherein, The modified A2 botulinum toxin contains one or more specific protease cleavage sites between positions 432 and 452, between positions 434 and 450, between positions 436 and 448, between positions 438 and 446, or between positions 440 and 444.

6. The modified type A2 botulinum toxin according to any one of claims 1 to 5, wherein, F1 and F2 are independently selected from GAGGAGS (SEQ ID NO:1), AGGS (SEQ ID NO:2), AGAG (SEQ ID NO:3), GSAGGS (SEQ ID NO:4), GSAGAG (SEQ ID NO:5), and GAGAG (SEQ ID NO:6); Optionally, F1 and F2 are independently selected from GAGGAGS (SEQ ID NO:1), AGGS (SEQ ID NO:2), and AGAG (SEQ ID NO:3).

7. The modified type A2 botulinum toxin according to any one of claims 1 to 6, wherein, F3 and F4 are independently selected from GAGGAGS (SEQ ID NO:1), AGGS (SEQ ID NO:2), AGAG (SEQ ID NO:3), GSAGGS (SEQ ID NO:4), GSAGAG (SEQ ID NO:5), GAGAG (SEQ ID NO:6), and AGAGKS (SEQ ID NO:7); Optionally, F3 and F4 are independently selected from GAGGAGS (SEQ ID NO:1), AGGS (SEQ ID NO:2), AGAG (SEQ ID NO:3), and AGAGKS (SEQ ID NO:7).

8. The modified type A2 botulinum toxin according to any one of claims 1 to 7, wherein, The specific protease cleavage sites are selected from: thrombin cleavage sites, 3C protease cleavage sites, TEV cleavage sites, PreScission protease cleavage sites, localizing enzyme cleavage sites, MMP-12 cleavage sites, MMP-13 cleavage sites, MMP-17 cleavage sites, MMP-20 cleavage sites, granzyme-B cleavage sites, enterokinase cleavage sites, SUMO protease cleavage sites, Factor Xa cleavage sites, and TVMV protease cleavage sites. Optionally, the specific protease cleavage site is selected from: thrombin cleavage site, 3C protease cleavage site, and TEV cleavage site.

9. The modified A2 type botulinum toxin according to claim 8, wherein, The amino acid sequence of the thrombin cleavage site includes LVPRGS (SEQ ID NO:8), LVPRGF (SEQ ID NO:9), or MYPRGN (SEQ ID NO:10), and optionally, the amino acid sequence of the thrombin cleavage site includes LVPRGS (SEQ ID NO:8). The amino acid sequence of the 3C protease cleavage site includes LEVLFQGP (SEQ ID NO:11); The amino acid sequence of the TEV cleavage site includes ENLYFQG (SEQ ID NO:12) and ENLYFQS (SEQ ID NO:13), and optionally, the amino acid sequence of the TEV cleavage site includes ENLYFQG (SEQ ID NO:12). The amino acid sequence of the enterokinase cleavage site includes DDDDK (SEQ ID NO:14); The amino acid sequence of the SUMO protease cleavage site includes EQIGG (SEQ ID NO:15); The amino acid sequence of the Factor Xa cleavage site includes IEGR (SEQ ID NO:16) and IDGR (SEQ ID NO:17); The amino acid sequence of the TVMV protease cleavage site includes ETVRFQS (SEQ ID NO:18).

10. The modified type A2 botulinum toxin according to any one of claims 1 to 9, wherein, The modified A2 type botulinum toxin contains one or more of the following amino acid sequences between cysteine ​​residues at positions 430 and 454: (1) KGAGGAGSLVPRGSAGGSA (SEQ ID NO: 19); (2) KAGAGKSLVPRGSAGAGA (SEQ ID NO: 20); (3) KGAGGAGSLVPRGSAGAGA (SEQ ID NO: 21); (4) KAGAGKSLVPRGSAGGSA (SEQ ID NO: 22); (5) KGAGGAGSLVPRGFAGGSA (SEQ ID NO: 23); (6) KAGAGKSLVPRGFAGAGA (SEQ ID NO: 24); (7) KGAGGAGSLVPRGFAGAGA (SEQ ID NO: 25); (8) KAGAGKSLVPRGFAGGSA (SEQ ID NO: 26); (9) KGAGGAGSMYPRGNAGGSA (SEQ ID NO: 27); (10) KAGAGKSMYPRGNAGAGA (SEQ ID NO: 28); (11) KGAGGAGSMYPRGNAGAGA (SEQ ID NO: 29); (12) KAGAGKSMYPRGNAGGSA (SEQ ID NO: 30); (13) KGAGGAGSLEVLFQGPAGGSA (SEQ ID NO: 31); (14) KAGAGKSLEVLFQGPAGAGA (SEQ ID NO: 32); (15) KGAGGAGSLEVLFQGPAGAGA (SEQ ID NO: 33); (16) KAGAGKSLEVLFQGPAGGSA (SEQ ID NO: 34); (17) KGAGGAGSENLYFQGAGGSA (SEQ ID NO: 35); (18) KAGAGKSENLYFQGAGAGA (SEQ ID NO: 36); (19) KGAGGAGSENLYFQGAGAGA (SEQ ID NO: 37); (20) KAGAGKSENLYFQGAGGSA (SEQ ID NO: 38); Optionally, the modified A2 botulinum toxin contains one or more of the following between cysteine ​​residues: (1) KGAGGAGSLVPRGSAGGSA (SEQ ID NO:19), (2) KAGAGKSLVPRGSAGAGA (SEQ ID NO:20), (14) KAGAGKSLEVLFQGPAGAGA (SEQ ID NO:32), and (18) KAGAGKSENLYFQGAGAGA (SEQ ID NO:36).

11. The modified type A2 botulinum toxin according to any one of claims 1 to 10, wherein, The modified A2 botulinum toxin further comprises one or more amino acid mutations, which do not cause the modified A2 botulinum toxin to inhibit neuronal activity in mammals and / or reduce muscle strength in mammals. This reduction in activity is relative to the amino acid sequences of NCBI ID:WP_012703873.1 (SEQ ID NO:87), NCBI ID:ABC26002 (SEQ ID NO:102), NCBI ID:ADB85243 (SEQ ID NO:98), NCBI ID:ADN04908 (SEQ ID NO:105), NCBI ID:AFM77673.1 (SEQ ID NO:100), NCBI ID:AFV13855.1 (SEQ ID NO:99), NCBI ID:AIT75847.1 (SEQ ID NO:97), and NCBI ID:AJE13255 (SEQ ID NO:99). Regarding the corresponding activities of the amino acid sequences of NCBI ID: KEI94032.1 (SEQ ID NO:101), NCBI ID: KEI94982 (SEQ ID NO:103), NCBI ID: NFD83166.1 (SEQ ID NO:110), NCBI ID: NFJ22948.1 (SEQ ID NO:107), NCBI ID: WP_079007110 (SEQ ID NO:106), NCBI ID: WP_205607627 (SEQ ID NO:109), and / or NCBI ID: WP_228605784 (SEQ ID NO:108), NCBI ID: 104, NCBI ID: KEI94032.1 (SEQ ID NO:101), NCBI ID: KEI94982 (SEQ ID NO:103), NCBI ID: NFD83166.1 (SEQ ID NO:110), NCBI ID: NFJ22948.1 (SEQ ID NO:107), NCBI ID: WP_079007110 (SEQ ID NO:106), NCBI ID: WP_205607627 (SEQ ID NO:109), and / or NCBI ID: WP_228605784 (SEQ ID NO:108).

12. The modified type A2 botulinum toxin according to any one of claims 1 to 11, wherein, The modified type A2 botulinum toxin contains an affinity tag at the N-terminus and / or C-terminus, the affinity tag including one or more of His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Soffag 1, Softag 3, TC, V5, VSV, Xpress, Halo, and Fc; Optionally, the affinity label includes His6; Alternatively, the affinity tag may comprise an amino acid sequence as shown in SEQ ID NO:

96.

13. The modified type A2 botulinum toxin according to any one of claims 1 to 12, wherein, The modified A2 type botulinum toxin does not contain affinity tags at either the N-terminus or the C-terminus. The affinity tags include one or more of His6, GST, Avi, Strep, S, MBP, Sumo, FLAG, HA, Myc, SBP, E, calmodulin, Soffag 1, Softag3, TC, V5, VSV, Xpress, Halo, and Fc.

14. The modified type A2 botulinum toxin according to any one of claims 1 to 12, wherein, The modified type A2 botulinum toxin has the following amino acid sequences: NCBI ID:WP_012703873.1 (SEQ ID NO:87), NCBI ID:ABC26002 (SEQ ID NO:102), NCBI ID:ADB85243 (SEQ ID NO:98), NCBI ID:ADN04908 (SEQ ID NO:105), NCBI ID:AFM77673.1 (SEQ ID NO:100), NCBI ID:AFV13855.1 (SEQ ID NO:99), NCBI ID:AIT75847.1 (SEQ ID NO:97), NCBI ID:AJE13255 (SEQ ID NO:104), NCBI ID:KEI94032.1 (SEQ ID NO:101), and NCBI ID:KEI94982 (SEQ ID NO:87). The amino acid sequences of NCBI ID: 103, NCBI ID: NFD83166.1 (SEQ ID NO: 110), NCBI ID: NFJ22948.1 (SEQ ID NO: 107), NCBI ID: WP_079007110 (SEQ ID NO: 106), NCBI ID: WP_205607627 (SEQ ID NO: 109), and / or NCBI ID: WP_228605784 (SEQ ID NO: 108) contain one or more of the specific protease cleavage sites between cysteine ​​residues at positions 430 and 454.

15. The modified type A2 botulinum toxin according to any one of claims 1 to 14, wherein, The modified type A2 botulinum toxin comprises any of the amino acid sequences shown in SEQ ID NOs:39-86, 88-95, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity with any of the amino acid sequences shown in SEQ ID NOs:39-86, 88-95.

16. The modified type A2 botulinum toxin according to any one of claims 1 to 15, wherein, The modified A2 type botulinum toxin can inhibit neuronal activity in mammals; Optionally, the mammal includes humans or rodents, such as rats or mice.

17. The modified type A2 botulinum toxin according to any one of claims 1 to 16, wherein, The modified A2 botulinum toxin can reduce muscle strength in mammals; Optionally, the mammal includes humans or rodents, such as rats or mice.

18. A nucleic acid molecule encoding the modified A2 type botulinum toxin according to any one of claims 1 to 17.

19. An expression vector comprising the nucleic acid molecule of claim 18.

20. A host cell comprising the nucleic acid molecule of claim 18 or the expression vector of claim 19; The host cells are selected from Escherichia coli cells, Bacillus subtilis cells, yeast cells, insect cells, and mammalian cells; Optionally, the host cell is Escherichia coli.

21. A method for preparing modified A2 botulinum toxin, comprising culturing the host cells of claim 20 under conditions favorable for expressing the modified A2 botulinum toxin of any one of claims 1 to 17.

22. A method for producing soluble double-stranded type A2 botulinum toxin, the method comprising: Step (1): Contact the modified type A2 botulinum toxin according to any one of claims 1 to 17 or the modified type A2 botulinum toxin prepared by the preparation method according to claim 21 with a specific protease capable of enzymatically cleaving the specific protease cleavage site.

23. The method according to claim 22, wherein, The method further includes: Step (2): Contact the product of step (1) with a hydrophobic surface to separate and obtain the soluble double-stranded type A2 botulinum toxin.

24. The method according to claim 23, wherein, The hydrophobic surface comprises an inert matrix with ligands composed of aryl or alkyl groups attached.

25. The method according to claim 24, wherein, The ligand is selected from butyl, phenyl, or octyl ligands.

26. A composition comprising soluble double-stranded type A2 botulinum toxin prepared by any one of claims 22 to 25.

27. The composition according to claim 26, wherein, The composition is a liquid or lyophilized powder composition.

28. The composition according to claim 26 or 27, wherein, The composition contains less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of single-chain type A2 botulinum toxin, based on the total weight of the composition.

29. Use of the modified A2 botulinum toxin according to any one of claims 1 to 17, the modified A2 botulinum toxin prepared by the preparation method according to claim 21, the soluble double-stranded A2 botulinum toxin prepared by the preparation method according to any one of claims 22 to 25, or the composition according to any one of claims 26 to 28 in the preparation of a cosmetic product.

30. Use of the modified A2 botulinum toxin of any one of claims 1 to 17, the modified A2 botulinum toxin prepared by the preparation method of claim 21, the soluble double-stranded A2 botulinum toxin prepared by the preparation method of any one of claims 22 to 25, or the composition of any one of claims 26 to 28 in the preparation of a medicament for reducing neuronal activity.

31. The use according to claim 30, wherein, The drug is used to treat one or more of the following: upper limb spasm, lower limb spasm, post-stroke spasm, spontaneous spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, orofacial dysphonia, tongue dystonia, neck dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasm and other voice disorders, spastic colitis, neurogenic bladder, anal spasm, limb spasm, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, pain caused by muscle spasm, skin conditions, tremor, migraine, psoriasis, allergic reactions, erythrocytic lymphohistiocytosis, alcoholic pancreatic disease, and glabellar lines.

32. A method for treating, preventing, or alleviating a disease, symptom, or disorder, said method comprising: The therapeutically effective amount of any one of claims 1 to 17, the modified type A2 botulinum toxin prepared by the preparation method of claim 21, the soluble double-stranded type A2 botulinum toxin prepared by the preparation method of any one of claims 22 to 25, or the composition of any one of claims 26 to 28 is administered to a subject in need.

33. The method according to claim 32, wherein, The diseases, symptoms, or disorders mentioned include one or more of the following: upper limb spasticity, lower limb spasticity, post-stroke spasticity, spontaneous spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, orofacial dysphonia, tongue dystonia, neck dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasm and other voice disorders, spastic colitis, neurogenic bladder, anal spasm, limb spasticity, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretion, pain caused by muscle spasms, dermatological conditions, tremor, migraine, psoriasis, allergic reactions, erythrocytic lymphohistiocytosis, alcoholic pancreatic disease, and glabellar lines.

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