Botulinum toxin type a mutants and uses thereof

Abstract

The application belongs to the technical field of medicine, and relates to a botulinum toxin type A mutant and application thereof. A series of botulinum toxin type A mutants are obtained by using a trinucleotide primer containing NNK at a mutation site, adopting site-directed mutagenesis technology to establish a single-site saturated mutation library and a combined mutation library, and screening. Compared with wild-type botulinum toxin type A, the botulinum toxin type A mutant has improved affinity with a receptor SV2C. The obtained full-length mutant toxin has improved activity of cutting a substrate SNAP-25 and muscle paralysis ability, reduced diffusion, significantly reduced lethal dose to mice, and improved safety, and can be applied to preparation of full-length BoNT / A mutant drugs for treating related diseases that can be treated by wild-type toxin and medical cosmetic related products. Meanwhile, the botulinum toxin type A mutant can be used as a drug carrier for drug delivery to the nervous system or an SV2 receptor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a botulinum toxin type A mutant and application thereof. The corresponding amino acid mutation sites of the mutant are located in the heavy chain receptor binding domain. BACKGROUND

[0002] Botulinum neurotoxins (BoNTs) are bacterial proteins produced by the anaerobic Clostridium botulinum and are considered to be the most potent toxins known. Seven different BoNT serotypes (A-G) have been discovered, each of which has different subtypes, such as BoNT / A1- / A10, BoNT / B1- / B8, BoNT / E1- / E12 and BoNT / F1- / F9. BoNTs are composed of a heavy chain (HC) and a light chain (LC) connected by a disulfide bond. The carboxyl-terminal of the heavy chain can recognize the cell surface receptor and mediate the entry of BoNT into neurons through receptor-mediated endocytosis. After entering the cell, when the endosome matures into a lysosome, the reduced pH (pH<4.5) will induce a conformational change in the amino-terminal region of the HC to form a pore, and then the catalytically active LC is delivered into the cell membrane. The LC is a highly specific metalloprotease that can cleave SNARE proteins, which can assemble into a complex composed of Syntaxin, SNAP-25 and Synaptobrevin (also known as VAMP2). The SNARE complex is essential for the fusion of synaptic vesicles with the cell membrane, thereby releasing neurotransmitters into the synaptic cleft. LC cleavage of SNARE proteins prevents this membrane fusion, thereby blocking nerve transmission, which in turn leads to the flaccid paralysis of botulism.

[0003] In 1989, the U.S. Food and Drug Administration (FDA) approved BoNT / A for the treatment of nervous system diseases, including eyelid spasm, strabismus and cervical dystonia. At present, the BoNT / A used in clinical practice at home and abroad is BoNT / A1. The indications of BoNT / A have been continuously expanded to muscle-skeletal diseases (such as muscle spasm, cerebral palsy), nerve damage (such as post-stroke spasm, spasm related to spinal cord injury) and chronic pain (such as migraine, tension headache, cluster headache). In addition, BoNT / A is also widely used in the field of cosmetic medicine. BoNT / A binds to the ganglioside (GBS) on the surface of neurons and its specific receptor synaptophysin SV2C, internalizes into the cell and specifically cleaves the substrate SNAP-25, thereby inhibiting the release of synaptic vesicles.

[0004] In view of the increasing wide medical applications of BoNT / A in clinical and medical cosmetology, BoNT / A is facing severe challenges. The toxin has a spreading effect when used in clinical applications. BoNT / A only needs to be injected into a certain muscle to cause varying degrees of muscle weakness in adjacent and distant areas. Especially, when a larger dose of BoNT / A is needed to be applied, the side effects of systemic toxicity caused by this spreading effect are more obvious. In addition, as the toxin spreading is recognized by the immune system, some patients may develop neutralizing antibodies after long-term use of BoNT / A, resulting in decreased or even lost efficacy. These limitations and adverse reactions seriously affect the overall effect of toxin therapy.

[0005] Enhancing the affinity between BoNT / A and its specific receptor SV2C through genetic engineering technology is a practical method to solve the problem of toxin diffusion and improve efficacy. Tao et al. (2017) used the bacterial adenylate cyclase two-hybrid (BACTH) system to screen the mutant library of Hc / B heavy chain receptor binding domain of type B botulinum toxin, and obtained the Hc / B mutant which improved the affinity with the receptor h-SytII. The inhibition of nerve conduction was increased by 11 times compared with wild-type BoNT / B HC. Studies have shown that the potency of BoNT / A4 is 1000 times lower than that of BoNT / A1. There is no significant difference in the ability of the two subtypes of light chain (LC) to catalyze the cleavage of SNAP-25. After mutating different amino acids in the receptor binding domain of BoNT / A4 to the same amino acids in BoNT / A1, the cleavage activity of BoNT / A4 can be restored to the level comparable to that of BoNT / A1. There is evidence that BoNT / A2 shows more obvious neuron toxicity than BoNT / A1, which is because BoNT / A2 enters the cell faster and more efficiently, regardless of its binding to GBS. These results suggest that the interaction between the Hc / A heavy chain receptor binding domain and the receptor SV2C is the key to determining the efficacy of BoNT / A. Benoit et al. showed that the key amino acids of the Hc / A of BoNT / A and the binding interface of SV2C are S1142, M1144, T1145, T1146, Y1149, R1156 and R1294, but after mutating the above-mentioned sites T1145 and T1146, BoNT / A no longer binds to its receptor SV2C, and after mutating R1156 and R1294, the binding ability is partially lost, which instead leads to the loss or reduction of the activity of the mutant. The dilemma of amino acid mutation of the Hc / A receptor binding domain of BoNT / A is that the currently used BoNT / A1 in clinical applications is the highest and longest-acting toxin subtype among all types and subtypes of botulinum toxin, and there is very limited space to further improve its biological activity and efficacy. Compared with wild-type BoNT / A, the biological activity and efficacy of the Hc / A receptor binding domain mutants disclosed in the prior art are significantly reduced.

[0006] CN202211112222.3 discloses that mutating Cys at positions 134 and 165 of the light chain, Cys at positions 791, 967 and 1060 of the heavy chain of BoNT / A1 reduces the probability of intra-chain disulfide bond mismatch. The mutant BoNT / A1 is 2.6 times more toxic than the wild type. Compared with the wild type, the mutant BoNT / A1 significantly increases the lethality of mice and enhances toxicity. Moreover, the preparation of BoNT / A1 in the patent application adopts separate expression of heavy chain and light chain, both of which form inclusion bodies and need to be denatured. The disulfide bonds are reduced during the denaturation process. Then, the refolding process is experienced, during which inter-chain disulfide bonds and intra-chain disulfide bonds are reformed. The wild type heavy chain and light chain have multiple Cys sites, and the refolded disulfide bonds are prone to mismatch, making it difficult to obtain BoNT / A1 with the desired performance. Therefore, the problem solved by the invention patent is that the incorrect structure of the product caused by disulfide bond mismatch during the purification process of the inclusion body form of BoNT / A1 reduces its activity. Obviously, the biological toxicity of the mutant disclosed in the invention patent is suitable for the preparation process of the heavy chain and the light chain expressed in the form of inclusion bodies, and the applicability of the complete wild type BoNT / A1 is low when soluble expression is performed in Clostridium botulinum or other suitable receptors or host cells. SUMMARY

[0007] To solve the above technical problems, the present application constructs a saturation mutation library for key amino acid residues in the receptor binding domain of BoNT / A1, and screens mutants that can enhance the affinity of BoNT / A1 and the receptor SV2C using the BACTH system. The mutant provided by the present application significantly improves the ability to cleave SNAP-25 in primary cultured rat hippocampal neurons of fetal mice. In addition, compared with the wild type BoNT / A1, the mutant described in the present application exhibits higher activity, lower systemic toxicity and higher safety when performing mouse lethality bioassay (MLB), digit abduction score (DAS) and toxin diffusion test. The BoNT / A1 mutant provided by the present application can be applied in the preparation of BoNT / A1 related drugs and medical and beauty related products for treating related diseases.

[0008] Specifically includes the following contents:

[0009] In a first aspect, the present application provides a mutant of A type botulinum toxin, the mutation site being in the receptor binding domain of the heavy chain. The mutant is selected from the following:

[0010] (1) any one of the following mutated amino acids: S1142F, S1142V, S1142Q, S1142I, S1142H;

[0011] and / or,

[0012] (2) any one of the amino acids of the mutations M1144L, M1144V, M1144W, M1144I;

[0013] and / or,

[0014] (3) the amino acid of the mutation T1146S.

[0015] Preferably, the amino acid sequence of the wild-type botulinum toxin type A BoNT / Al is as shown in SEQ ID NO. 1 and the genetic sequence is as shown in SEQ ID NO. 4.

[0016] Preferably, the mutant is selected from any one of the amino acids of the mutations S1142I, S1142V, S1142Q, S1142F, M1144L, M1144I, M1144V, T1146S.

[0017] Preferably, the mutant is selected from any one of the amino acids of the mutations S1142I, S1142V, S1142Q, S1142F, M1144L, M1144I, M1144V, T1146S.

[0018] S1142F and M1144I;

[0019] S1142I and M1144V;

[0020] S1142V and M1144V;

[0021] S1142Q and M1144V;

[0022] S1142F and M1144V;

[0023] S1142I and M1144L;

[0024] S1142V and M1144L;

[0025] S1142Q and M1144L;

[0026] S1142F and M1144L;

[0027] S1142H and M1144W.

[0028] Preferably, the mutant is selected from any one of the amino acids of the mutations S1142I, S1142V, S1142Q, S1142F, M1144L, M1144I, M1144V, T1146S.

[0029] S1142V and M1144V;

[0030] S1142V and M1144L;

[0031] S1142F and M1144L;

[0032] S1142H and M1144W.

[0033] Preferably, the mutant is an amino acid mutated at S1142F and M1144L.

[0034] Preferably, the mutant has an amino acid sequence as set forth in SEQ ID NO. 2.

[0035] Preferably, the mutant is an amino acid mutated at S1142F and M1144L.

[0036] S1142V, M1144I and T1146S;

[0037] S1142Q, M1144I and T1146S;

[0038] S1142V, M1144V and T1146S;

[0039] S1142V, M1144L and T1146S;

[0040] S1142F, M1144L and T1146T;

[0041] S1142H, M1144W and T1146S.

[0042] Preferably, the mutant is an amino acid mutated at S1142F and M1144L.

[0043] S1142V, M1144L and T1146S;

[0044] S1142F, M1144L and T1146T;

[0045] Preferably, the mutant is an amino acid mutated at S1142V, M1144L and T1146S.

[0046] Preferably, the mutant has an amino acid sequence as set forth in SEQ ID NO. 3.

[0047] In a second aspect, the present application provides use of the botulinum toxin type A mutant of the first aspect in the preparation of a therapeutic drug.

[0048] Preferably, the therapeutic drug comprises at least one of improving / treating blepharospasm, strabismus, spastic dystonia, migraine, tumor.

[0049] In a third aspect, the present application provides use of the botulinum toxin type A mutant of the first aspect in the preparation of a medical cosmeceutical product.

[0050] Preferably, the medical cosmeceutical product comprises at least one of improving moderate to severe glabellar lines, wrinkle removal, face slimming, leg slimming.

[0051] In a fourth aspect, the present application provides use of the botulinum toxin type A mutant of the first aspect in the manufacture of a drug targeted delivery carrier.

[0052] Preferably, the drug targeted delivery carrier comprises a nervous system or SV2C receptor drug targeted delivery carrier.

[0053] In a fifth aspect, the present application provides a pharmaceutical composition comprising the botulinum toxin type A mutant of the first aspect and a pharmaceutically acceptable excipient.

[0054] In a sixth aspect, the present application provides use of the pharmaceutical composition of the fifth aspect in the manufacture of a therapeutic drug.

[0055] Preferably, the therapeutic drug comprises at least one of improving / treating blepharospasm, strabismus, spastic dystonia, migraine, and tumor.

[0056] In a seventh aspect, the present application provides use of the pharmaceutical composition of the fifth aspect in the manufacture of a medical cosmeceutical product.

[0057] Preferably, the medical cosmeceutical product comprises at least one of improving moderate-to-severe glabellar lines, wrinkle removal, face slimming, and leg slimming.

[0058] The present application has the following beneficial effects: the present application uses a trinucleotide primer containing NNK at a mutation site, uses site-directed mutagenesis technology to establish a single-site saturated mutation library and a combinatorial mutation library, and screens to obtain a series of botulinum toxin type A heavy chain receptor binding domain mutants; the affinity of the botulinum toxin type A heavy chain receptor binding domain mutant to the receptor SV2C is improved; replacing the wild-type Hc / A in the full-length toxin, the obtained full-length mutant toxin has improved activity in cutting substrates SNAP-25 and muscle paralysis ability, reduced diffusion, reduced toxicity, and improved safety, and can be used in the preparation of full-length BoNT / A mutant drugs for treating related diseases that can be treated by wild-type toxins and medical cosmeceutical products. At the same time, the BoNT / A mutant can be used as a drug carrier for targeted delivery of nervous system or SV2C receptor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 Hc / A mutants identified in the BACTH assay were further analyzed by β-galactosidase activity, which reflects the level of recombinant adenylyl cyclase (Student's t-test, n = 4, *P < 0.05, ns indicates P > 0.05); error bars are shown as mean ± SEM.

[0060] Figure 2Comparison of β-galactosidase activity of the combined mutants of Hc / A; the vertical axis represents the relative value of β-galactosidase activity of each mutant relative to wt, and the horizontal axis represents each mutant; P>0.05, no significance, and P<0.05, significance, for FITS compared with wt; error bars are shown as mean ± SEM, n=4.

[0061] Figure 3 SDS-PAGE results of purification of full-length toxin; Fig. 1: BoNT / A wt; 2: FLTT; 3: VLTS.

[0062] Figure 4 Results of activity detection of full-length A1 mutant botulinum toxin at cellular level; a is the result of Western Blot analysis, the toxin was incubated with hippocampal neurons for 48 h, and 0, 7.5, 15, 31.25, 62.5 and 125 pM of toxin were used for incubation; b is the semi-quantitative analysis result of a, using gray value as the measurement index, the horizontal axis is the concentration of toxin, and the vertical axis is the proportion of cleaved SNAP-25 to total SNAP-25, all data shown are mean ± SEM (n=4); c is the semi-quantitative comparison result of cleavage of SNAP-25 by wt, FLTT and VLTS when the concentration of toxin is 31.25 pM.

[0063] Figure 5 Results of mouse toe spread score (DAS) experiment; each dose (pg / mouse) was converted to its natural logarithm, and the highest average DAS score (mean DAS max) of each dose group was plotted. Data were curve-fitted with a four-parameter Logistic equation, with the lower asymptote constrained to 0 and the upper asymptote constrained to 4; the dose value obtained from the equation corresponds to half of the maximum DAS (DAS2), which was converted to ED50 by inverting the calculated value in the natural logarithm scale to its linear value. 50 .

[0064] Figure 6 Concentration-response curve of percent body weight change; %BW is represented as the difference between the most obvious day of body weight change (day 2) and the weight before injection, divided by the weight before injection; the concentration-response curve of %BW and toxin concentration was obtained by linear fitting, and all data are represented as mean (n=4).

[0065] Figure 7Toxin diffusion area was measured by fluorescent imaging in mice; A, fluorescent toxin was injected into the back muscle, and the diffusion area was observed by live animal imaging system at 8 h; B, the fluorescent area was measured by Image J software, and the average value was obtained by repeating the measurement 3 times for each image, (Student's t-test, n = 3, *P < 0.05), and the error bar represents SEM.

[0066] Figure 8 Survival curves of mice injected with 25 U of BoNT / Awt, BoNT / A-FLTT and BoNT / A-VLTS in the gastrocnemius muscle of the left hind limb; ED 50 1 U was defined. DETAILED DESCRIPTION

[0067] The present application uses a BACTH system kit (bacterial adenylate cyclase-based two-hybrid, BACTH) to screen Hc / A mutants. The system is a two-hybrid method based on the reconstruction of adenylate cyclase in E. coli. Studies have shown that the catalytic domain of adenylate cyclase (CyaA) is divided into two subdomains: a 25 kDa fragment containing the catalytic site (T25, residues 1-224) and an 18 kDa fragment containing the calmodulin binding site (T18, residues 225-399). T25 and T18 are not active when physically separated. When they are fused to interacting polypeptides X and Y, respectively, the interaction of X and Y leads to functional complementation of the two fragments of T25 and T18, restoring the activity of adenylate cyclase. The reconstructed adenylate cyclase can catalyze the production of cAMP, which in turn forms a cAMP / CAP complex, positively regulates the expression of the structural genes of the lac operon of lactose catabolism, and therefore, X and Y have an interaction, which can produce blue colonies on the medium containing X-Gal substrate, and the darker the color, the stronger the interaction between X and Y. If there is no interaction between the two, the colonies are white. In addition, the interaction between X and Y can be further quantified by measuring the level of cAMP or the enzyme activity of β-galactosidase.

[0068] The present application utilizes the trinucleotide primer containing NNK at the mutation site, adopts the site-directed mutagenesis technology to establish the single site saturation mutation library for the 1142, 1144, 1145, 1146, 1149, 1156 and 1294 sites of Hc / A1, and constructs the combined mutation library for 1142 / 1144, 1145 / 1146 and 1142 / 1144 / 1145 / 1146. The T18-Hc / A1 mu of each mutation library is co-transformed with T25-SV2C-L4 into BTH101 chemically competent, and is grown on the LB agar medium containing X-Gal. In theory, when the library and SV2C-L4 are at very low concentration and the competent amount is sufficient, the recombinant vector T18-Hc / A1 containing a mutant will enter a competent cell. If the Hc / A1 mutant interacts with SV2C-L4, the colony is blue. If the Hc / A1 mutant does not combine or combines weakly with SV2C-L4, the colony is white. The deep blue colony is picked and cultured, and then the plasmid with higher activity than the control group (T18-Hc / A1 wt and T25-SV2C-L4) is selected for DNA sequencing. If mutation occurs at the corresponding site, the β-galactosidase activity of the mutant is determined separately.

[0069] The surface plasmon resonance technology (SPR) is adopted to determine the affinity of the candidate mutant, and the biological activity and pharmacodynamics of the mutant BoNT / A1 obtained through screening are evaluated at the cell and animal levels. Meanwhile, the wild type BoNT / A1 is used as a control for comparison.

[0070] The present application screens six candidate mutants S1142F / M1144L, S1142F / M1144L / T1146S, S1142V / M1144L, S1142V / M1144L / T1146S, S1142V / M1144V and S1142H / M1144W with significantly improved β-galactosidase activity through the BACTH method, and further determines the affinity of the candidate mutant and the receptor SV2C-L4 by using the SPR technology. The results show that the affinity of each mutant and SV2C-L4 is improved by different degrees compared with Hc / Awt and SV2C-L4, and the mutants S1142F / M1144L and S1142V / M1144L / T1146S are improved by 6.3 and 5.6 times, respectively, compared with Hc / Awt.

[0071] The application introduces mutant S1142F / M1144L and S1142V / M1144L / T1146S into BoNT / A1 full-length by site-directed mutagenesis technology. Cell level enzyme cutting activity detection is carried out on the obtained mutants, and their cutting activity on substrate SNAP-25 is evaluated, and the results show that the cutting activity of mutants S1142F / M1144L and S1142V / M1144L / T1146S on SNAP-25 is significantly better than that of wild type BoNT / A1. Animal level activity detection is carried out on the obtained mutants, and their lethality, activity, diffusion and safety are evaluated through MLB experiment, DAS experiment and toxin diffusion experiment, and the results show that the mutants S1142F / M1144L and S1142V / M1144L / T1146S have higher muscle paralysis activity, lower lethality, lower diffusion and higher safety than the wild type.

[0072] The amino acid sequence of wild type A type botulinum toxin BoNT / A1 of the application is shown as SEQ ID NO. 1, and the gene sequence is shown as SEQ ID NO. 4; the amino acid sequence of the S1142F / M1144L mutant (FLTT) is shown as SEQ ID NO. 2; the amino acid sequence of the S1142V / M1144L / T1146S mutant (VLTS) is shown as SEQ ID NO. 3; and the gene sequence of the SV2C-L4 is shown as SEQ ID NO. 5.

[0073] The technical solutions of the application are described in further detail below in combination with specific examples, but the protection scope of the application is not limited to the following examples.

[0074] Example 1 Construction of forward and reverse saturation mutation library

[0075] 1.1 Biological materials

[0076] BACTH system kit (item number: EUK001, purchased from Euromedex company, http: / / www.euromedex.com), which includes pUT18C (T18) vector, pKT25 (T25) vector and recipient cell BTH101. Recombinant plasmid T18-Hc / A and recombinant plasmid T25-SV2C-L4 are synthesized by GenScript, and the gene sequence of SV2C-L4 is shown as Seq ID No: 5. Hc / A and SV2C-L4 are respectively connected to the BamHI and EcoRI sites of T18 and the BamHI and EcoRI sites of T25. Top10 chemically competent, DH5α electroporation competent, and BTH101 chemically competent are prepared by the conventional method of preparing electrocompetent cells in the laboratory.

[0077] 1.2 Reagents

[0078] 2xHieff Gold PCR Master Mix high-fidelity enzyme premix (Shanghai Yisen Biotechnology Co., Ltd., Item No. 10149ES03);

[0079] DpnI methylated template digestion enzyme (NEB, Item No. R0176);

[0080] 5-bromo-4-chloro-3-indolyl-D galactoside (X-Gal) (Shanghai Sangon Biological Engineering Co., Ltd., Item No. A600083-0001);

[0081] o-nitrophenyl-β-D-galactopyranoside (ONPG) (Beijing Solabio Technology Co., Ltd., Item No. O8040);

[0082] β-mercapto reducing agent (Beijing Solabio Technology Co., Ltd., M8210).

[0083] 1.3 Implementation steps

[0084] 1.3.1 Construction of forward saturation mutation library

[0085] (1) NNK primer design

[0086] Using recombinant plasmid T18-Hc / A1 as a template, NNK primers were designed at S1142, M1144, T1145, T1146, Y1149, R1156 and R1294 sites, including primer sequences for constructing unit point saturation mutation library and primer sequences for constructing combined site saturation mutation library. SM is a combined mutation library of S1142 and M1144 sites, TT is a combined mutation library of T1145 and T1146 sites, and SMTT is a combined mutation library of S1142, M1144, T1145 and T1146 sites, as shown in Table 1.

[0087] Table 1 NNK primer sequences for constructing saturation mutation library

[0088]

[0089] (2) Site-directed mutagenesis

[0090] First, mix the DNA template with the NNK primer, amplify it through the PCR program, then degrade the original template using DpnI enzyme, and then concentrate and purify the PCR product by sodium acetate ethanol precipitation method. The specific steps are as follows:

[0091] 1) PCR amplification: The PCR reaction system was configured on ice, 1 μL of DNA template, 2.5 μL of forward primer (10 μM), 2.5 μL of reverse primer (10 μM), and 25 μL of 2x PCR high-fidelity enzyme premix were added, and ddH2O was added to make the total system 50 μL. The pre-denaturation-denaturation-reannealing-extension-termination extension program was cycled, as shown in Table 2.

[0092] Table 2 PCR amplification program for site-directed mutagenesis

[0093]

[0094] 2) Template degradation: After PCR amplification, the PCR product was placed on ice for 2 min, and then 2 μL of DpnI enzyme, 6 μL of 10x Cutsmart buffer, and 2 μL of ddH2O were added to make a 60 μL enzyme digestion system with the PCR product. It was placed at 37°C for 4-8 h.

[0095] 3) Product purification:

[0096] ① 6 μL of sodium acetate (3M, pH 5.2) was added to the DNA solution and mixed well;

[0097] ② 130 μL of ice-precooled ethanol was added, and after mixing well, it was placed in a -20°C refrigerator for 30 min;

[0098] ③ 12,000 g centrifugation for 5 min, carefully remove the supernatant, and suck off all the liquid drops on the wall of the tube;

[0099] ④ 750 μL of 70% ethanol was added, and 12,000 g centrifugation was performed for 2 min. The supernatant was carefully removed, and all the liquid drops on the wall of the tube were sucked off;

[0100] ⑤ The open EP tube was placed on the clean bench at room temperature and blown for 8-10 min until there was no ethanol smell.

[0101] ⑥ 10 μL of ddH2O was added to dissolve the DNA precipitate.

[0102] (3) Electroporation

[0103] Take 2 μL library DNA and 40 μL DH5α competent cells, mix well, and carefully transfer to the pre-cooled shock cup (note that no bubbles are generated), place on ice for use; set the parameters for electric transformation as 25 μF, 200 Ω, 1.8 KV, and perform electric transformation. After electric transformation, add 1 mL of SOC liquid medium to the tank, mix well by blowing and sucking, transfer the bacterial solution to a 10 mL EP tube, add 1 mL of SOC liquid medium, mix well, and then place in a constant temperature shaking incubator at 37°C and 250 rpm for 1 h of recovery. Spread 2 mL of bacterial solution evenly on a 25 cm LB agar plate, and place in a constant temperature incubator at 37°C for 12-16 h of stationary culture.

[0104] (4) Library extraction

[0105] Add 8-10 mL of LB liquid medium to the petri dish covered with colonies, gently scrape the colonies with a sterile spatula and transfer to a 50 mL centrifuge tube. Centrifuge at 10,000 rpm for 3 min, discard the supernatant, and extract the bacterial bodies according to the steps of the plasmid extraction kit to obtain the mutant library and store it in a -20°C refrigerator for standby use.

[0106] (5) Sequencing verification

[0107] Before library extraction, a portion of single colonies is picked and cultured in LB liquid medium, and then the plasmid is extracted and sent to the sequencing department of Shengong Biotechnology Co., Ltd. Xi'an for sequencing. The sequencing results need to have a certain diversity, so as to ensure the integrity of the library.

[0108] The library capacity of this example is measured by the number of colonies formed per unit mass of library plasmid after transformation, i.e., colony-forming units (CFU). Gradient dilution method is used to dilute the bacterial solution after transformation, spread on a 10 cm petri dish, and count after the colonies grow. For example, 1 ng / μL SMTT library transformation, when diluted 100 times, the number of colonies is 45, and the library capacity = 45 x 100 CFU / 1 ng = 4.5 x 10 6 CFU / μg.

[0109] The library diversity in this example is evaluated by sequencing part of the library colonies, and the proportion of mutants in the total number of sequencing is used as the evaluation index. In theory, the mutant type also contains the wild type wt, which is ignored in the experiment. The experimental results show that the mutant proportion in T1145 library and T1146 library is less than 70%, indicating that the quality of these two libraries is not good, and the mutant proportion of the remaining libraries is higher than 80%, and the types of mutants are different, indicating that the diversity of these libraries is good.

[0110] 1.3.2 Construction of reverse saturation mutant library based on BACTH system screening

[0111] The above-mentioned saturated mutant library is screened by the BACTH system, and many mutants obtained by sequencing are still wild type, so it is suspected that this may be due to incomplete degradation of the template by DpnI enzyme, leading to this situation. In order to avoid the interference of wild type, we use the forward saturated mutant library to screen the inactive mutants of each mutation site by the BACTH system, and then use the inactive mutants as templates to construct the reverse saturated mutant library by NNK primers to exclude the interference of wild type.

[0112] (1) Obtaining of inactive mutants

[0113] The mutant library constructed with T18-Hc / A as a template is called a forward saturated mutant library. 1 μL of the forward saturated mutant library (1 ng / μL) is mixed with 1 μL of T25-SV2C-L4 (10 ng / μL), 100 μL of BTH101 chemically competent cells are added, mixed, placed in an ice bath for 30 min, heated in a 42°C metal bath for 90 s, and then ice-bathed again for 2 min. 200 μL of LB liquid medium is added, and the mixture is incubated in a constant temperature shaking incubator at 37°C and 250 rpm for 45 min. The bacterial solution is diluted to a certain degree and uniformly coated on LB / X-Gal screening medium, and incubated in a constant temperature incubator at 30°C for 48 h. Then, 3 white single colonies of each library are inoculated into a shaking tube containing 4 mL of LB liquid medium, and incubated in a constant temperature shaking incubator at 37°C and 250 rpm overnight. The next day, plasmid extraction is performed according to the plasmid extraction kit. The extracted plasmid is sent to the sequencing department of GenScript Biotech Corporation in Xi'an for sequencing. If there is a mutation at the library mutation site, it is considered to be correct.

[0114] Plasmid purification: The plasmid after sequencing is a mixture of T18-mutant and recombinant plasmid T25-SV2C-L4, so the mixed plasmid needs to be transformed into Top10 chemically competent cells by heat shock transformation. The transformation product is uniformly coated on LB agar medium containing 100 μg / mL Amp. T25-SV2C-L4 contains Kan resistance but no Amp resistance, so it cannot grow in LB agar medium containing Amp. After incubation in a constant temperature incubator at 37°C for 12-16 h, single colonies are inoculated into LB liquid medium, incubated in a constant temperature shaking incubator at 37°C and 250 rpm for 12-16 h, and then plasmid extraction is performed according to the plasmid extraction kit. The extracted plasmid is stored in a -20°C refrigerator.

[0115] After the plasmid was purified, the T18-mutant and T25-SV2C-L4 were co-transformed again into BTH101 chemically competent, and cultured in a constant temperature incubator at 30°C for 48 hours. If all the colonies were white, it was proved that the mutant was not active.

[0116] (2) Construction of reverse saturation mutant library

[0117] In line with the method of constructing the forward saturation mutant library, the only difference was that the PCR template was the inactive mutant plasmid extracted from the white colonies in the forward mutant library.

[0118] The inactive mutant was used as a template to construct a reverse saturation mutant library corresponding to the forward saturation mutant library to exclude the screening bias caused by wt residues. The library capacity and mutant proportion were used to evaluate the library quality. The results showed that the mutant proportion in the reverse library reached 100% due to the exclusion of the disadvantages of using wt as a template for library construction. The sequencing results from small-scale testing showed that all the mutants were different types, indicating that the reverse library had good diversity and uniformity.

[0119] 1.3.3 Protein interaction analysis based on BACTH system

[0120] Through multiple rounds of screening of the forward and reverse saturation mutant libraries by the BACTH system, blue colonies were selected for preliminary β-galactosidase activity determination, and plasmids with higher activity than wt were selected for sequencing to obtain the main mutation types at each site. Combined with the analysis of unit point saturation mutant library and combined mutant library, it was concluded that S1142, M1144 and T1146 were the active plastic regions, and the amino acid substitution types at S1142 site were mainly I / V / Q / F; the amino acid substitution types at M1144 site were mainly L / I / V; and the substitution at T1146 site was mainly T / S. T1145, Y1149, R1156 and R1294 were mainly wild type, and Y1149F and R1156W / S appeared at a lower frequency (shown in Table 3).

[0121] Further β-galactosidase activity analysis of the mutants S1142I / V / Q / F, M1144L / I / V, T1146S, Y1149F, R1156S / W identified in the BACTH test showed that the mutants S1142I / V / Q / F, M1144L / I / V and T1146 had strong β-galactosidase activity, indicating that the binding of these mutants to the receptor SV2C was enhanced. Figure 1

[0122] Table 3 Screening results of saturation mutant library ​

[0123]

[0124]

[0125] Note: In letters like LLTS, the letters represent the amino acid substitution types at positions S1142, M1144, T1145, and T1146, respectively. wt indicates wild type, and "-" indicates that no active mutants were selected.

[0126] Based on the screening results of the forward saturation mutant library and the reverse saturation mutant library, the amino acid variable regions are concentrated at the S1142 and M1144 sites, S1142I / V / Q / F, M1144I / L / V, there are a total of 12 combinations of pairwise mutations, and when combined with T1146S, there will be 24 mutants. We obtained a subset of mutants, FLTT (S1142F / M1144L), VLTT (S1142V / M1144L / T1146S), IVTT (S1142I / M1144V), VVTT (S1142V / M1144V), QVTT (S1142Q / M1144V), FITT (S1142F / M1144I), and HWTT (S1142H / M1144W), through screening of combinatorial mutant libraries. Based on these, we constructed ILTT (S1142I / M1144L), VLTT (S1142V / M1144L), QLTT (S1142Q / M1144L), and IVTT (S1142I / M1144S) through site-directed mutagenesis. Combination mutants including 4V), HWTS(S1142H / M1144W / T1146S), FLTS(S1142F / M1144L / T1146S), FVTT(S1142F / M1144V), IITS(S1142I / M1144I / T1146S), VITS(S1142V / M1144I / T1146S), QITS(S1142Q / M1144I / T1146S), FITS(S1142F / M1144I / T1146S), VVTS(S1142V / M1144V / T1146S), and FVTS(S1142F / M1144V / T1146S) were successfully constructed through sequencing verification.

[0127] Comparison of β-galactosidase activity between various combined mutants and wt showed a significant increase in all cases. Figure 2 (As shown). Among them, mutants such as VVTT, VLTT, VLTS, FLTT, FLTS, and HWTT have relatively high activity, so these mutants were selected for subsequent purification and used for the next step of in vitro affinity determination.

[0128] Example 2 Determination of the affinity of Hc / A mutants to SV2C-L4 using surface plasmon resonance technology

[0129] 2.1 Biomaterials

[0130] Hc / A1 was recombined between Nde I and EcoR I sites of pET28a(+) vector to obtain pET 28a(+)-Hc / A1 recombinant plasmid, which was used to prepare His6-Hc / A1 wild type and mutant proteins. Mutant recombinant plasmids were prepared using the Multi-point Site-directed Mutagenesis Kit (Item No. 11004ES10) of Jinsheng Biological Company according to the manufacturer's instructions.

[0131] The SV2C-L4 sequence was synthesized by Jinweizhi Company, and the fragment was cloned between BamHI and EcoRI sites of pGEX-4T-1 vector to obtain the protein expression plasmid pGEX-4T-1-SV2C-L4, which was used to prepare GST-SV2C-L4.

[0132] BL21(DE3) and DH5α chemically competent cells were obtained from Shenguo Bioengineering Company.

[0133] The pET 28a(+)-Hc / A1 wt and each mu mutant recombinant vectors that were successfully constructed and verified by sequencing were transformed into BL21(DE3) competent cells using the conventional heat shock transformation method, and the pGEX-4T-1-SV2C-L4 recombinant vector was transformed into DH5α competent cells using the conventional heat shock transformation method. Single colonies were picked one by one and inoculated in 20 mL of LB medium containing 80 μg / mL Kan, and 100 μg / mL Amp was added to the LB medium for inoculating pGEX-4T-1-SV2C-L4. The seed was prepared by incubating at 37°C, 200 rpm overnight. The above bacterial solution was inoculated in 500 mL of LB medium containing the appropriate antibiotic at 5%, and incubated at 37°C, 200 rpm until the OD 600 was 0.6, and then 0.2 mM IPTG was added to induce expression for 5 hours. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min.

[0134] The above bacteria were added to 20 mM PB pH 7.4 buffer at a ratio of 1:30, the bacterial cells were broken by ultrasonic, and the solution was clear. Centrifugation at 10,000 rpm for 10 min, and the supernatant was taken. His6-Hc / A1 wt and each mutant were purified by affinity chromatography using Ni Sepharose 6FF filler from Cytiva, and GST-SV2C-L4 was purified by affinity chromatography using Glutathione Sepharose 4FF filler from Cytiva. The purification was performed according to the manufacturer's instructions. The purified His6-Hc / A1 wt and each mutant and GST-SV2C-L4 were dialyzed against 20 mM PB pH 7.4 overnight, and the protein concentrations were determined by BCA method. Among them, His6-Hc / A1 wt and each mutant were 7 μg / mL for VLTS and 50 μg / mL for others, and GST-SV2C-L4 was 100 μg / mL. The above protein solutions were aliquoted and stored at -20°C.

[0135] 2.2 Reagents

[0136] Series S Sensor Chip CM5 (GE, USA, item number 29149603);

[0137] Amine Coupling Kit (GE, USA, BR100633);

[0138] Glycine 2.0 (GE, USA, BR100355);

[0139] HBS-EP+10x (GE, USA, BR100669).

[0140] 2.3 Implementation steps

[0141] (1) Instrument preparation

[0142] Start the Biacore T200 control software, place the HBS-EP+ buffer and deionized water on the corresponding cradle, connect the corresponding pipeline, open the chip cabin door after the instrument runs stably, install the CM5 chip, and set the relevant information. The detection temperature of the sample flow cell is set to 25°C.

[0143] (2) GST-SV2C-L4 pH detection

[0144] Before immobilizing the ligand on the CM5 chip, the pH of the ligand buffer needs to be screened so that the ligand can be enriched near the chip surface by electrostatic adsorption, and better coupling effect can be achieved. Usually, Acetate 4.0, Acetate 4.5, Acetate 5.0 are screened; GST-SV2C-L4 is diluted to 5 ng / μL with Acetate 4.0, Acetate 4.5, Acetate 5.0, respectively, the binding time is 180 s, the flow rate is 10 μL / min, and then the chip surface is regenerated with 50 mM NaOH.

[0145] (3) GST-SV2C-L4 coupling

[0146] Mix 0.4M EDC and 0.1M NHS in the amino coupling kit at a ratio of 1:1, flow through the chip surface Fc2 at a flow rate of 10 μL / min, and activate for 200 s to activate the carboxymethyl dextran matrix on the chip surface; dilute GST-SV2C-L4 to 5 ng / μL with Acetate 4.0, flow through the activated chip surface at a flow rate of 10 μL / min, and covalently couple GST-SV2C-L4 to the chip surface, with a target coupling amount of 200 RU; block the activated sites on the chip surface with ethanolamine, and block for 200 s.

[0147] Activate and block the chip surface Fc1 using the same procedure as the reference channel.

[0148] (4) Kinetic analysis

[0149] Dilute Hc / Awt, S1142H / M1144W (HWTT) to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 μM, and 7.8125 nM with HBS-EP+buffer as repeatability for repeated concentration test; dilute S1142V / M1144L (VLTT), S1142V / M1144L / T1146S (VLTS), S1142F / M1144L (FLTT), S1142F / M1144L / T1146S (FLTS), and S1142V / M1144V (VVTT) to 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 μM, 3.90625 nM, 1.953125 nM, and 1.953125 nM with HBS-EP+buffer as repeatability for repeated concentration test.

[0150] Different concentrations of protein ligands were flowed over the chip surface at a flow rate of 30 μL / min, the binding time was set to 60 s, the dissociation time was set to 60 s, the regeneration reagent was selected as Glycine 2.0, and the regeneration time was 30 s; after the kinetic program was run, the kinetic results were fitted by using the Biacore T200 analysis software. The kinetic parameters of the binding of S1142F / M1144L, S1142V / M1144L / T1146S and SV2C-L4 are shown in Table 4.

[0151] Table 4 Kinetic parameters of the interaction between GST-SV2C and Hc / A mutants in the SPR assay

[0152] Hc / A k on (1 / Ms)]]> k off (1 / s) KD (nM) Rmax (RU) wt 5.319E+5 0.04537 85.29 49.04 FLTT 1.031E+6 0.01405 13.62 50.84 VLTS 9.211E+5 0.01404 15.24 58.50 VLTT 7.162E+5 0.01126 15.73 63.46 FLTS 6.447E+5 0.01478 22.92 46.23 VVTT 6.379E+5 0.01657 25.98 37.09 HWTT 9.404E+5 0.04917 52.29 45.60

[0153] The KD values in Table 4 indicate the affinity of the mutants to the receptor SV2C, and it can be seen that the affinity of the Hc / A mutants to the extracellular domain of the receptor obtained by screening in the application is in the order of FLTT > VLTS > VLTT > FLTS > VVTT > HWTT > wt. Among them, FLTT has the strongest affinity to the receptor SV2C, which is 6.3 times the affinity of WT to the receptor SV2C; followed by VLTS, which is 5.6 times the affinity of wt.

[0154] Example 3 Cell level detection of the enzymatic activity of BoNT / A mutants on substrate SNAP-25

[0155] 3.1 Biological materials

[0156] BoNT / A1 wt and BoNT / A1-FLTT (FLTT) and BoNT / A1-VLTS (VLTS) were prepared according to the method of Miyashita et al. (Miyashita SI, Zhang J, Zhang S, et al., Science translational medicine, 2021, 13(575): eaaz4197). Among them, the mutant recombinant vector was obtained by site-directed mutagenesis of BoNT / A1 wt using the multi-point site-directed mutagenesis kit of Yixing Biotech, and the SDS-PAGE results of the target protein after purification are shown in Figure 3 ; SD pregnant mice (SPF level) at 18 days of pregnancy were from Lanzhou Animal Research Institute, license number SCXK(Gan)2020-0002.

[0157] 3.2 Reagents

[0158] SNAP-25 antibody (GeneTex, item number GTX113839);

[0159] β-actin antibody (Immunoway, item number YM3028);

[0160] RIPA lysis buffer (Solarbio, Cat# R0010);

[0161] Neurobasal™ Medium (Thermo Fisher Scientific, Cat# 21103-049);

[0162] B-27 Supplement (Thermo Fisher Scientific, Cat# 17504-044);

[0163] GlutaMAX™-1 (Thermo Fisher Scientific, Cat# 35050-061).

[0164] 3.3 Implementation steps

[0165] (1) Extraction of hippocampal neurons

[0166] Preparation: Polylysine-coated coverslips, 0.2M HC1-washed, 75% ethanol-sterilized coverslips were placed in a 24-well plate, 0.1 mg / mL polylysine was added to each well (100-200 μL), and the plate was incubated overnight at room temperature. The polylysine was aspirated and the coverslips were washed with sterile water three times and blown dry for later use.

[0167] Isolation of hippocampus: pregnant mice were sacrificed with high concentration of CO2, the embryos were removed, the heads of the fetal mice were cut off, and the heads were placed in a culture dish containing dissection solution; transferred to a clean bench, observed under a dissecting microscope to separate the hippocampus, placed in a centrifuge tube containing dissection solution, and placed in an ice box; aspirate the dissection solution, add 0.05% preheated trypsin, incubate in a 37°C water bath, shake every 10 minutes; aspirate the trypsin, add 10 mL of pre-cooled dissection solution each time, invert the centrifuge tube 5 times, and wash 3 times; aspirate the dissection solution, add 1 mL of neuron culture medium, blow 8 times against the bottom of the centrifuge tube to disperse the block-shaped tissue into single cells, and avoid air bubbles; supplement the corresponding volume of neuron culture medium, invert the centrifuge tube to disperse the cells evenly, 600 μL per well, and inoculate in a 24-well plate coated with polylysine; incubate in a 37°C incubator for 12 days for subsequent experiments.

[0168] (2) Western Blot

[0169] BoNT / Awt, along with mutants FLTT and VLTS, were diluted with neuronal culture medium to create a series of concentrations: 7.5 pM, 15 pM, 31.25 pM, 62.5 pM, and 125 pM. Corresponding concentrations of botulinum toxin were added to neurons in 24-well plates, mixed, and incubated at 37°C for 48 h. 70 μL of RIPA lysis buffer was added to each well, and cells were lysed at 4°C for 10 min. Cells were then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was added to 5×SDS-PAGE protein loading buffer, and the cells were denatured at 100°C for 10 min. SDS-PAGE electrophoresis conditions were: 80 V for 30 min, followed by 120 V for 1.5 h. Appropriately sized PVDF membranes were cut, activated in methanol for 20 s, and then bonded together with the PVDF membrane in a cellulose pad-filter paper-PVDF membrane-gel-filter paper-cellulose pad configuration. Transfer was performed at 300 mA for 2 h. After transfer, a 5% skim milk powder solution was prepared using TBST buffer as the blocking buffer and blocked at room temperature for 2 hours. The primary antibody was diluted 1:5,000 and incubated overnight at 4°C. The membrane was washed three times with TBST buffer at 60 rpm for 10 minutes each time. The secondary antibody was diluted 1:10,000 and incubated at room temperature for 1 hour. The membrane was washed three times with TBST buffer at 60 rpm for 10 minutes each time. Protein bands were developed using ECL chemiluminescence buffer, and images were recorded and photographed using a gel imaging system.

[0170] The results of BoNT / A1 wt, FLTT, and VLTS are as follows: Figure 4 As shown. Figure 4 The results showed that mutants FLTT and VLTS exhibited significant SNAP-25 cleavage at a low concentration of 7.5 pM, while BoNT / A1 wt only showed SNAP-25 cleavage at 15 pM. The lower the toxin concentration corresponding to SNAP-25 cleavage, the stronger the toxin's effect; therefore, mutants FLTT and VLTS showed stronger SNAP-25 cleavage efficacy than wt. Western blot analysis, including grayscale measurements and a line graph showing the percentage of cleaved SNAP-25 versus toxin concentration, further visually demonstrated that mutants FLTT and VLTS had superior SNAP-25 cleavage efficacy compared to BoNT / A1 wt. Figure 4 (As shown in b and c).

[0171] Example 4: Animal-based study of the activity, lethality, and spread of the BoNT / A mutant.

[0172] 4.1 Biomaterials

[0173] BoNT / A1 wt, FLTT, and VLTS are wild-type or mutant full-length toxins prepared in Example 3.

[0174] Clean level Kunming mice were purchased from Lanzhou University Experimental Animal Center, license number SCXK(Gan)2023-0003.

[0175] 4.2 Implementation steps

[0176] 4.2.1 BoNT / A1 wt and its mutants based on mouse lethal bioassay (MLB) study

[0177] In this study, clean level 18-22 g female Kunming mice were used to detect the biological activity of BoNT / A1 wt and its mutants FLTT and VLTS. First, the wt and its mutants FLTT and VLTS were diluted with PBS containing 0.25% BSA to contain 75 pg, 37.5 pg, 18.75 pg, 9.375 pg of toxin per 100 μL. Then the mice were randomly divided into 5 groups, 4 mice in each group, and each mouse was injected intraperitoneally with 100 μL of wt and its mutants FLTT and VLTS, and the survival of mice was recorded within 4 days. In addition, wt and FLTT were diluted to contain 120 pg, 105 pg, 97.5 pg, 90 pg, 82.5 pg and 75 pg of toxin per 10 μL, and VLTS was diluted to contain 210 pg, 180 pg, 165 pg, 150 pg and 135 pg of toxin per 10 μL, and then the mice were randomly divided into groups, 4 mice in each group, and each mouse was injected with 10 μL of left hind leg gastrocnemius muscle, and the survival of mice was recorded within 4 days.

[0178] 4.2.2 BoNT / A wt and its mutants based on mouse DAS score functional study

[0179] The foot abduction score DAS is a physiological model for assessing muscle relaxation. This model is based on the fact that when mice are briefly suspended by the tail, they exhibit a typical startle response characterized by hind limb extension and foot abduction. This model is on a 5-point scale (0-4), with a DAS value of 0 indicating complete abduction, while a DAS value of 4 indicates no abduction and leg extension, indicating complete muscle paralysis.

[0180] In the MLB test study, the LD 50 On this basis, a gradient concentration was set, with 60 pg, 30 pg, 15 pg, 5 pg, 1 pg, 0.5 pg of each dose group injected, with a volume of 10 μL. The mice were randomly divided into 3 groups, and each group was randomly assigned to 6 dose groups. Each mouse was weighed and labeled one day before injection. The gastrocnemius muscle of the left hind leg of the mouse was injected. Within 12 h of injection, DAS score was recorded every 2 h, then every 12 h or once a day, and the body weight of the mouse was recorded daily.

[0181] 4.2.3 Fluorescence imaging experiment of mice

[0182] Ni 2+ Hc / Awt and its heavy chain receptor binding domain mutants FLTT and VLTS were purified by affinity chromatography. Then, the dialysis was exchanged with 0.1 M NaHC03, after which 10 μL SF680 fluorescent dye was added per milliliter of protein and incubated at room temperature for 3 hours. Finally, the dialysis was exchanged with PBS buffer. After the back of the mice was shaved, 2 μL of Hc / Awt and mutants Hc / AFLTT and VLTS were injected into the back muscle. Then, fluorescence imaging was performed at 0 hours and 8 hours, respectively, using a small animal living imaging system. The statistical fluorescence area was calculated using Image J software.

[0183] 4.2 Implementation results

[0184] The results of the MLB experiment, DAS score experiment, and toxin receptor binding domain diffusion experiment are shown in Figure 5 to Figure 7 and Table 5.

[0185] Table 5. Results of effectiveness, safety, and diffusion related tests of BoNT / A mutant toxins in vivo

[0186] BoNT / A1 ED 50 (pg) 0% BW (pg) 0% BW / ED 50 ]] [ip LD 50 (pg)]]> im LD 50 (pg)]]> wt 5.76 24.40 4.24 21.08 107.2 FLTT 4.66 25.59 5.49 33.18 112.4 VLTS 4.05 65.84 16.25 33.18 168.7

[0187] The intraperitoneal median lethal dose ip LD 50 reflects the lethal activity of the toxin, and the smaller the value, the stronger the lethal activity of the toxin. According to the ip LD 50 The results show that wt < FLTT = VLTS, which indicates that the lethal activity of mutants FLTT and VLTS is lower than that of wt (Table 5). The diffusion of the toxin from the injection site in the gastrocnemius muscle to other sites such as the respiratory muscle can induce systemic toxicity and cause the animal to die. The intramuscular median lethal dose im LD 50 to some extent reflects the diffusion of the toxin, and the larger the value, the lower the diffusion of the toxin. According to the im LD 50 Results: VLTS > FLTT > wt, indicating that the diffusion of mutants VLTS and FLTT is significantly lower than that of wt (Table 5).

[0188] The DAS scores of mice showed obvious dose dependence, and the DAS scores of each dose group reached a maximum value around 1 day after administration, and the peak value generally lasted for 2-3 days, after which the DAS scores began to decrease, indicating that the muscle paralysis of mice was recovering. According to the corresponding highest average DAS scores of each dose group, a non-linear regression curve was drawn, and according to the corresponding toxin dosage when the DAS was 2, the ED 50 of BoNT / A1 wt and its mutants FLTT and VLTS was obtained after logarithmic conversion. 50The value size relationship is: VLTS < FLTT < wt Figure 5 and Table 5), indicating that the muscle paralysis ability of mutants FLTT and VLTS is superior to wt.

[0189] 0% body weight change (%BW) represents the dose of toxin that does not cause any decrease in body weight, and thus reflects the degree of systemic diffusion of the toxin from the injection site. From the 0% BW results, wt < FLTT < VLTS, suggesting that mutants FLTT and VLTS show lower diffusion than wt ( Figure 6 ).

[0190] While the ED 50 between 0% BW represents the safety range of the toxin, which is a key pharmacological parameter. FLTT and VLTS show slightly better ED 50 than wt, and have higher 0% BW values. Among them, the main difference of VLTS is 0% BW: up to about 65.84 pg can be injected without causing weight loss, while wt can only be injected about 24.40 pg wt. Therefore, the safety of VLTS is 3.83 times that of wt (16.25 vs 4.24, Table 5).

[0191] In the DAS score experiment, the 0% BW value of the mouse is im LD 50 analysis, the diffusion of mutants FLTT and VLTS is lower than that of wt. In order to further measure the diffusion range of muscle injection of the toxin, we purified Hc / Awt and Hc / AFLTT and VLTS, and used a small animal live imaging system to measure the diffusion area after a period of time after injection of the toxin. It was found that the measured fluorescent area of VLTS (0.394 ± 0.045 cm 2 ) and the measured fluorescent area of FLTT (0.470 ± 0.045 cm 2 ) were significantly lower than the measured fluorescent area of wt (0.561 ± 0.039 cm 2 ), indicating that the diffusion of mutants FLTT and VLTS is lower than that of wt ( Figure 7 ).

[0192] The mutants BoNT / A-FLTT and BoNT / A-VLTS prepared by the present application have lower diffusion and higher safety than the wild type, and the ED 50 is improved. Defining the ED 50 as 1 U, 25 U of BoNT / Awt, BoNT / A-FLTT and BoNT / A-VLTS were respectively given to the mice for injection into the gastrocnemius muscle of the left hind limb, and it was found that at 5 days after injection, all BoNT / A wt mice died, 62.5% of the BoNT / A-FLTT group survived, and all BoNT / A-VLTS survived.Figure 8 )。

[0193] The deficiency of the clinical application of the BoNT / A1 product mainly surrounds the side effects on the human body caused by the diffusion at the injection site, and the poor or ineffective treatment effect caused by the neutralizing antibodies in some people. The present application is directed to the modification of the BoNT / A1 receptor binding domain Hc / A1, so as to improve the binding ability of Hc / A1 to the receptor SV2C (SV2C-L4 is the extracellular domain part of SV2C), increase the ability of the toxin heavy chain HC to mediate the light chain LC of the toxin to enter the target cell, reduce the diffusion of the toxin, thereby improving the therapeutic effect, reducing the amount of toxin, and further reducing the generation of neutralizing antibodies.

[0194] The present application utilizes the BACTH system to screen the binding domain related sites of Hc / A1 and SV2C-L4 for saturation mutation, and determines the in vitro affinity constant of part of the screened mutants. By recombining the obtained Hc / A1 mutants into the full-length toxin of BoNT / A1, the influence of the Hc / A1 mutation modification on the activity of BoNT / A1 is evaluated at the level of hippocampal neuron cells and whole animals of mice.

[0195] The present application utilizes the BACTH system to screen the key amino acid residues of the binding domain of Hc / A1 and SV2C-L4 for saturation mutation, and determines the superior amino acids and the transformable sites of the sites. Among them, S1142, M1144 and T1146 are the active plastic regions, and suitable substitution can improve the affinity of HC / A and the receptor SV2C, wherein the activity is higher when S1142 is substituted for I / V / Q / F, the amino acid change at M1144 position is more concentrated, and mainly substituted for I / L / V or W. The T1146 position is mainly replaced by S, but in individual mutants, it can lead to loss of activity. T1145, Y1149, R1156 and R1294 are the active non-plastic regions, and it is not recommended to screen the mutations of these 4 sites of Hc / A1.

[0196] The present application further determines the β-galactosidase activity and SPR affinity of several active mutants obtained by screening, and determines 2 mutant combinations with improved activity compared with wt, wherein the affinity of FLTT to SV2C is about 6.3 times higher than that of wt, and the affinity of VLTS to SV2C is about 5.6 times higher than that of wt.

[0197] Based on the SNAP-25 cleavage activity detection of the whole botulinum toxin process at the cell level, the cutting ability of the full-length mutants FLTT and VLTS of BoNT / A1 to SNAP-25 is obviously higher than that of BoNT / A1 wt.

[0198] The mouse lethal bioactivity assay, mouse DAS score activity assay and mouse fluorescence imaging analysis results of the BoNT / A1 mutant of the application show that the muscle paralysis ability of full-length BoNT / A1 mutants FLTT and VLTS is better than that of BoNT / A1 wt, and at the same time has lower diffusion and lethality.

[0199] Starting from the BoNT / A1 mutant amino acid sequence disclosed in the application, the nucleotide sequence thereof can be deduced by using a codon table, and after full synthesis by using a codon optimization method, the BoNT / A1 mutant gene sequence is more suitable for expression in different host cells, but does not affect the primary structure of the expression product protein amino acid, which is understood by those skilled in the art.

[0200] The application discloses that the S1142, M1144 and T1146 at the receptor binding interface of the mutant BoNT / A1 heavy chain receptor binding domain are replaced or not replaced into FLT or VLS. Accordingly, the heavy chain of other subtypes or other toxins of SV2 as the receptor and subtypes thereof are subjected to sequence alignment, and the same mutation is performed at the homologous and conserved sites as the amino acid mutation sites in the application, so that the diffusion of the toxin is reduced, and the safety thereof is improved, which can be understood by those skilled in the art.

[0201] BoNT / A1 can be used to treat a very wide range of diseases. BoNT / A1 was first used in ophthalmology to treat strabismus in the late 1970s, and currently, the indications mainly include blepharospasm, spastic dystonia, chronic migraine and the like; in dermatology, BoNT / A1 is used to temporarily improve moderate-to-severe glabellar lines, wrinkle removal and face and leg slimming, treatment of hyperhidrosis and prevention of surgical scars and the like. In recent years, the application potential of BoNT / A1 in the treatment of depression and tumors has gradually emerged. In addition, BoNT / A1 can also be used in neurology: spastic torticollis, hemifacial spasm and the like; rehabilitation treatment of nerve injury: spinal cord injury, neurogenic bladder and brain trauma; acting on parasympathetic synapses: excessive secretion of sweat glands (hyperhidrosis, polydipsia), bromidrosis; causing muscle spasm and the like; inhibiting peripheral trigeminal vascular system peptide release: treating migraine and toothache; other: cardiospasm, various sphincter spasm, constipation and the like. BoNT / A1 product Botox was on the cover of Time magazine on January 16, 2017, and it was claimed that it can also be used for the treatment of 793 other diseases.

[0202] Botulinum neurotoxin BoNTs can specifically enter motor neurons to cause botulism. Inactivation of BoNT / A and its nanobodies can be fused to its nanobodies, and inactivated BoNT / A can specifically deliver its nanobodies into poisoned neurons, thereby playing the role of nanobody antidote (Sci Transl Med. 2021; 13(575): eaaz4197). In addition, since neuroendocrine tumors highly express the receptors of BoNT / A, small molecule toxins such as auristatin, maytansine and its analogs, calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxin and quinoline alkaloid (SN-38) can be used as delivery carriers to deliver small molecule toxins into tumors and kill tumor cells efficiently (Biochimie 2010; 92(9): 1252-9; Cancer Gene Therapy 2020; 27: 898-909). BoNT / A itself can also be used as an anti-tumor application (Prostate 2009; 69: 1143-1150; Int J Clin Exp Pathol 2015; 8: 8411-8418). Obviously, the mutant disclosed in the present application is more efficient than its wild type as a delivery carrier in the form of Hc / A, whole toxin or mutant inactivated whole toxin, which can be understood by those skilled in the art.

[0203] In summary, the beneficial effects of the present application are that the Hc / A of BoNT / A is mutated to improve its affinity to the receptor SV2C. Replacing the wild type Hc / A in the full length toxin, its activity of cutting substrate SNAP-25 and muscle paralysis ability are improved, the diffusion is reduced, and the safety is improved. The mutant of type A botulinum toxin disclosed in the present application is added with pharmaceutically acceptable buffer ingredients, salts and other auxiliary ingredients to form a pharmaceutical composition, which can be used in the preparation of drugs or related medical and beauty products for treating diseases that can be treated by wild type toxin. At the same time, the BoNT / A mutant can be used as a drug carrier for targeted delivery of nervous system drugs.

Claims

1. A type A botulinum toxin mutant, characterized in that, Compared with the wild-type botulinum toxin type A BoNT / A1 shown in SEQ ID NO.1, the mutant is composed of amino acids with mutations in S1142F and M1144L; the amino acid sequence of the mutant is shown in SEQ ID NO.

2.

2. A type A botulinum toxin mutant, characterized in that, Compared with the wild-type botulinum toxin type A BoNT / A1 shown in SEQ ID NO.1, the mutant is composed of amino acid mutations of S1142V, M1144L and T1146S; the amino acid sequence of the mutant is shown in SEQ ID NO.

3.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the type A botulinum toxin mutant as described in any one of claims 1-2 and pharmaceutically acceptable excipients.

4. The use of the type A botulinum toxin mutant as described in any one of claims 1-2 or the composition as described in claim 3 in the preparation of therapeutic drugs, medical aesthetic products, or drug targeted delivery carriers; The therapeutic drug is for improving / treating at least one of blepharospasm, strabismus, spastic dystonia, and migraine; The medical aesthetic product is used to improve at least one of moderate to severe frown lines, wrinkle removal, face slimming, and leg slimming. The drug delivery carrier includes a delivery carrier for drugs targeting the nervous system or SV2C receptors.

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