Botulinum toxin A mutant and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a botulinum toxin A mutant and application thereof. A trinucleotide primer containing NNK at a mutation site is utilized, a site-specific mutagenesis technology is adopted to establish a single-site saturated mutation library and a combined mutation library, and a series of A-type botulinum toxin mutants are obtained through screening; compared with wild type A botulinum toxin, the affinity of the A botulinum toxin mutant and a receptor SV2C is improved; wild Hc / A in the full-length toxin is replaced, the activity and the muscle paralysis causing capacity of the obtained full-length mutant toxin cutting substrate SNAP-25 are improved, the diffusivity is reduced, the lethal dose to mice is remarkably reduced, the safety is improved, and the toxicity of the whole-length mutant toxin cutting substrate SNAP-25 is reduced. The method can be applied to preparation of drugs for treating related diseases which can be treated by wild type toxins through full-length BoNT / A mutant drugs and medical cosmetology related products. Meanwhile, the botulinum toxin A mutant can be used as a drug carrier for targeted nervous system or SV2 receptor drug delivery.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a botulinum toxin type A mutant and an application thereof, wherein the corresponding amino acid mutation sites of the mutant are all located in the heavy chain receptor binding domain. Background Art

[0002] Botulinum neurotoxins (BoNTs) are bacterial proteins produced by anaerobic Clostridium botulinum and are considered to be the most potent toxins known. Seven different BoNT serotypes (AG) have been discovered, each with different subtypes, such as BoNT / A1- / A10, BoNT / B1- / B8, BoNT / E1- / E12, and BoNT / F1- / F9. BoNTs consist of a heavy chain (HC) and a light chain (LC) linked by disulfide bonds. The carboxyl terminus of the heavy chain can recognize cell surface receptors, mediating the entry of BoNTs into neurons through receptor-mediated endocytosis. Once inside the cell, when the endosome matures into a lysosome, the reduced pH (pH < 4.5) induces conformational changes in the amino-terminal region of the HC to form a pore, which then delivers the catalytically active LC into the cell membrane. LC is a highly specific metalloprotease that cleaves SNARE proteins, which assemble into a complex consisting 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 neurotransmission and causing the flaccid paralysis of botulism.

[0003] In 1989, the U.S. Food and Drug Administration (FDA) approved BoNT / A for the treatment of neurological diseases, including blepharospasm, strabismus, and cervical dystonia. At present, the BoNT / A used clinically at home and abroad is BoNT / A1. The indications of BoNT / A have been continuously expanded to musculoskeletal diseases (such as muscle spasms, cerebral palsy), nerve damage (such as post-stroke spasms, spinal cord injury-related spasms) 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 gangliosides (GBS) on the surface of neurons and their specific receptor synaptic vesicle protein SV2C, is internalized into cells and specifically cleaves the substrate SNAP-25, thereby inhibiting the release of synaptic vesicles.

[0004] Given its increasingly widespread medical applications in clinical and aesthetic medicine, BoNT / A faces severe challenges. The toxin has a diffusion effect when used clinically. BoNT / A only needs to be injected into a certain muscle to cause varying degrees of muscle weakness in adjacent and distant areas. In particular, when larger doses of BoNT / A are required, the side effects of systemic toxicity caused by this diffusion effect are more obvious. In addition, since the toxin diffuses and is recognized by the immune system, some patients may produce neutralizing antibodies after long-term use of BoNT / A, resulting in reduced or even loss of 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 way 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 the heavy chain receptor binding domain of botulinum toxin type B Hc / B. The obtained Hc / B mutant had improved affinity with the receptor h-SytII and increased the inhibitory effect on neurotransmission by 11 times compared with the 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 chains (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 a level comparable to that of BoNT / A1. There is evidence that BoNT / A2 shows more obvious neuronal toxicity than BoNT / A1, which is because BoNT / A2 enters cells faster and more efficiently, regardless of GBS binding. These results suggest that the interaction between the Hc / A heavy chain receptor binding domain and the receptor SV2C is key to determining the efficacy of BoNT / A. Benoit et al. showed that the key amino acids in the Hc / A and SV2C binding interface of BoNT / A are S1142, M1144, T1145, T1146, Y1149, R1156 and R1294. However, after mutating the above sites T1145 and T1146, BoNT / A no longer binds to its receptor SV2C, and after mutating R1156 and R1294, the binding ability of the two is partially lost, which leads to the loss or reduction of the activity of the mutant. The dilemma of carrying out amino acid mutations in the Hc / A receptor binding domain of BoNT / A is that the BoNT / A1 currently used in clinical practice is the most potent and longest-lasting toxin subtype among all types and subtypes of botulinum toxin, and there is very limited room for further improving 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] Chinese patent CN202211112222.3 discloses the mutation of Cys at positions 134 and 165 of the light chain of BoNT / A1, and Cys at positions 791, 967, and 1060 of the heavy chain, which reduces the probability of intrachain disulfide bond mismatch. The mutant BoNT / A1 is 2.6 times more toxic than its wild type. Compared with the wild type, the lethality of the mutated BoNT / A1 to mice is significantly increased, and the toxicity is enhanced. Moreover, the preparation of BoNT / A1 in this patent invention adopts the expression of heavy chain and light chain separately, both of which form inclusion bodies and need to be denatured separately, and the disulfide bonds are reduced during the denaturation process. It then undergoes renaturation, and the interchain disulfide bonds and intrachain disulfide bonds are re-formed during the renaturation process; there are Cys at multiple sites in the wild-type heavy chain and light chain, and the renatured disulfide bonds are prone to mismatch, making it difficult to obtain the expected BoNT / A1. Therefore, the problem solved by this invention patent is that the product structure is incorrect due to the mismatch of disulfide bonds during the expression and purification of BoNT / A1 in inclusion bodies, which reduces its activity. Obviously, the improved biological toxicity of the mutant disclosed in this invention patent is applicable to the preparation process when the specific heavy chain and light chain are expressed in inclusion bodies respectively, and has low applicability to the complete wild-type BoNT / A1 for soluble expression using botulinum toxin or other suitable receptors or host cells. Summary of the invention

[0007] In response to the above technical problems, the present invention constructs a saturation mutation library for key amino acid residues in the heavy chain receptor binding domain of botulinum neurotoxin type A1 (BoNT / A1), and uses the BACTH system to screen out mutants that can enhance the affinity of BoNT / A1 to the receptor SV2C. The mutants provided by the present invention significantly improve the cleavage ability of SNAP-25 in primary cultured rat fetal hippocampal neurons. In addition, compared with the wild-type BoNT / A1, the mutants described in the present invention showed higher activity, lower systemic toxicity and higher safety due to significantly reduced diffusivity in the mouse lethal bioassay (MLB), toe abduction score (DAS) and toxin diffusion test. The BoNT / A1 mutants provided by the present invention can be used in the preparation of drugs for related diseases that can be treated by BoNT / A1 and medical beauty-related products.

[0008] Specifically include the following:

[0009] In a first aspect, the present invention provides a botulinum toxin type A mutant, wherein the mutation site is in the heavy chain receptor binding domain, and compared with the wild-type botulinum toxin type A BoNT / A1, the mutant is selected from:

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

[0011] and / or,

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

[0013] and / or,

[0014] (3) Amino acid of T1146S mutation.

[0015] Preferably, the amino acid sequence of the wild-type botulinum toxin type A BoNT / A1 is shown as SEQ ID NO.1, and the gene sequence is shown as SEQ ID NO.4.

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

[0017] Preferably, the mutant is selected from any one of the following groups of mutated amino acids:

[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 following groups of mutated amino acids:

[0029] S1142V and M1144V;

[0030] S1142V and M1144L;

[0031] S1142F and M1144L;

[0032] S1142H and M1144W.

[0033] Preferably, the mutants are amino acids of S1142F and M1144L mutations.

[0034] Preferably, the amino acid sequence of the mutant is shown as SEQ ID NO.2.

[0035] Preferably, the mutant is selected from any one of the following groups of mutated amino acids:

[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 selected from any one of the following groups of mutated amino acids:

[0043] S1142V, M1144L and T1146S;

[0044] S1142F, M1144L and T1146T;

[0045] Preferably, the mutants are amino acids of S1142V, M1144L and T1146S mutations.

[0046] Preferably, the amino acid sequence of the mutant is shown in SEQ ID NO.3.

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

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

[0049] In a third aspect, the present invention provides the use of the botulinum toxin type A mutant described in the first aspect in the preparation of medical cosmetic products.

[0050] Preferably, the medical cosmetic product includes at least one of improving moderate to severe frown lines, removing wrinkles, slimming the face, and slimming the legs.

[0051] In a fourth aspect, the present invention provides use of the botulinum toxin type A mutant described in the first aspect in the preparation of a drug targeted delivery vector.

[0052] Preferably, the drug targeted delivery vector includes a targeted delivery vector for a nervous system or SV2C receptor drug.

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

[0054] In a sixth aspect, the present invention provides use of the pharmaceutical composition described in the fifth aspect in the preparation of therapeutic drugs.

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

[0056] In a seventh aspect, the present invention provides use of the pharmaceutical composition described in the fifth aspect in the preparation of medical cosmetic products.

[0057] Preferably, the medical cosmetic product includes at least one of improving moderate to severe frown lines, removing wrinkles, slimming the face, and slimming the legs.

[0058] The beneficial effects of the present invention are as follows: the present invention uses a trinucleotide primer containing NNK at the mutation site, adopts site-directed mutagenesis technology to establish a single-site saturation mutation library and a combinatorial mutation library, and screens and obtains a series of botulinum toxin type A heavy chain receptor binding domain mutants; the botulinum toxin type A heavy chain receptor binding domain mutant has improved affinity with the receptor SV2C; the full-length mutant toxin obtained by replacing the wild-type Hc / A in the full-length toxin has improved activity and muscle paralysis ability of the substrate SNAP-25, reduced diffusivity, reduced toxicity, and improved safety, and can be used in the preparation of full-length BoNT / A mutant drugs for the treatment of related diseases that can be treated by wild-type toxins and medical beauty-related products. At the same time, BoNT / A mutants can be used as drug carriers when delivering drugs targeting the nervous system or SV2C receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Hc / A mutants were analyzed by β-galactosidase activity; Hc / A mutants identified in the BACTH assay were further analyzed by β-galactosidase activity, which reflects the level of recombinant adenylate 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 Hc / A combined mutants; the vertical axis represents the relative value of β-galactosidase activity of each mutant relative to wt, and the horizontal axis represents each mutant; except for FITS compared with WT, P>0.05, which was not significant, the other mutants were compared with wt, P<0.05, which was significant; error bars were shown as mean±SEM, n=4.

[0061] Figure 3 SDS-PAGE results of the purified full-length toxin; in the figure, 1: BoNT / Awt; 2: FLTT; 3: VLTS.

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

[0063] Figure 5 Results of the mouse toe abduction score (DAS) experiment; each dose (pg / mouse) was converted to its natural logarithm, and the highest mean DAS score (mean DAS max) of each dose group was plotted. The data were fitted with a four-parameter logistic equation curve, 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 ED by inverting the calculated value in the natural logarithmic scale with its linear value 50 .

[0064] Figure 6 Concentration response curve of percentage change in body weight; %BW is expressed as the ratio of the difference between the body weight on the day with the most obvious body weight change (day 2) and the body weight before injection to the body weight before injection; the response curve of %BW and toxin concentration was obtained by linear fitting, and all data are expressed as mean values ​​(n=4).

[0065] Figure 7The diffusion area of ​​the toxin was determined by mouse fluorescence imaging; A was injected into the dorsal muscle with fluorescent toxin and observed using the small animal in vivo imaging system 8 hours later; B was measured using Image J software, and each image was measured three times and the average was taken (Student's t-test, n=3, *P<0.05), and the error bars represent SEM.

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

[0067] The present invention adopts the 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 Escherichia coli. Studies have shown that the catalytic domain of adenylate cyclase (CyaA) is divided into two subdomains: a 25kDa fragment containing a catalytic site (T25, residues 1-224) and an 18kDa fragment containing a calmodulin binding site (T18, residues 225-399), and T25 and T18 are inactive when physically separated. When the two are fused with interacting polypeptides X and Y respectively, the interaction between X and Y causes the two fragments T25 and T18 to produce functional complementarity, thereby restoring the activity of adenylate cyclase. The reconstructed adenylate cyclase can catalyze the production of cAMP, thereby forming a cAMP / CAP complex, which positively regulates the expression of the structural genes of the lac operon of lactose catabolism. Therefore, X and Y interact with each other and can produce blue colonies on a medium containing the X-Gal substrate. The darker the color, the stronger the interaction between X and Y. If there is no interaction between the two, the colonies will be white. In addition, the interaction between X and Y can be further quantified by measuring the cAMP level or the enzyme activity of β-galactosidase.

[0068] The present invention uses a trinucleotide primer containing NNK at the mutation site, and adopts site-directed mutagenesis technology to establish a single-site saturation mutation library at 1142, 1144, 1145, 1146, 1149, 1156, and 1294 sites of Hc / A1, and constructs a combination mutation library at 1142 / 1144, 1145 / 1146, and 1142 / 1144 / 1145 / 1146. Each mutant library T18-Hc / A1 mu is co-transformed with T25-SV2C-L4 into a BTH101 chemical competent state, and grown on an LB agar medium containing X-Gal. Theoretically, when the concentration of the library and SV2C-L4 is very low and the competent state dosage is sufficient, a mutant recombinant vector T18-Hc / A1 will enter a competent cell accordingly. If the Hc / A1 mutant interacts with SV2C-L4, the colony is blue. If the Hc / A1 mutant does not bind to SV2C-L4 or binds very weakly, the colonies will be white. Pick the dark blue colonies for culture, and then use the β-galactosidase activity assay to select plasmids with higher activity than the control group (T18-Hc / A1 wt and T25-SV2C-L4) for DNA sequencing. If a mutation occurs at the corresponding site, the β-galactosidase activity of the mutant is then assayed separately.

[0069] The surface plasmon resonance (SPR) technique was used to measure the affinity of candidate mutants, and the biological activity and efficacy of the mutant BoNT / A1 obtained by screening were evaluated at the cell and animal levels. At the same time, the wild-type BoNT / A1 was used as a control for comparative study.

[0070] The present invention screened and obtained 6 candidate mutants S1142F / M1144L, S1142F / M1144L / T1146S, S1142V / M1144L, S1142V / M1144L / T1146S, S1142V / M1144V and S1142H / M1144W with significantly improved β-galactosidase activity by BACTH method, and further measured the affinity of the candidate mutants to the receptor SV2C-L4 by SPR technology. The results showed that the affinity of each mutant to SV2C-L4 was improved to varying degrees compared with Hc / Awt and SV2C-L4, among which the affinity of mutants S1142F / M1144L and S1142V / M1144L / T1146S was improved by 6.3 and 5.6 times respectively compared with Hc / Awt.

[0071] The present invention uses site-directed mutagenesis technology to introduce mutations S1142F / M1144L and S1142V / M1144L / T1146S into the full length of BoNT / A1. The obtained mutants are subjected to cell-level enzymatic activity detection to evaluate their cleavage activity on the substrate SNAP-25. The results show that the cleavage activity of mutants S1142F / M1144L and S1142V / M1144L / T1146S on SNAP-25 is significantly better than that of wild-type BoNT / A1. The obtained mutants are also subjected to animal-level activity detection, and their lethality, activity, diffusibility and safety are evaluated through MLB experiments, DAS experiments and toxin diffusibility experiments. The results show that mutants S1142F / M1144L and S1142V / M1144L / T1146S have higher muscle paralysis activity, lower lethality, lower diffusibility and higher safety than the wild type.

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

[0073] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

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

[0075] 1.1 Biomaterials

[0076] BACTH system kit (Cat. No. EUK001, purchased from Euromedex, 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 were fully synthesized by GENEWISE, and the gene sequence of SV2C-L4 is shown in Seq ID No: 5. Hc / A and SV2C-L4 were connected to the BamHI and EcoRI sites of T18 and the BamHI and EcoRI sites of T25, respectively. Top10 chemical competent cells, DH5α electroporation competent cells, and BTH101 chemical competent cells were prepared in this laboratory according to the conventional method for preparing electroporation competent cells.

[0077] 1.2 Reagents

[0078] 2×Hieff Gold PCR Master Mix high-fidelity enzyme premix (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10149ES03);

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

[0080] 5-Bromo-4-chloro-3-indolyl-D-galactoside (X-Gal) (Shanghai Sangon Biotechnology Co., Ltd., Catalog No. A600083-0001);

[0081] o-Nitrophenyl-β-D-galactopyranoside (ONPG) (Beijing Solaibao Technology Co., Ltd., Catalog No. O8040);

[0082] β-Mercapto reducing agent (Beijing Solebow 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 the recombinant plasmid T18-Hc / A1 as a template, NNK primers were designed at the S1142, M1144, T1145, T1146, Y1149, R1156 and R1294 sites, including primer sequences for constructing a single-site saturation mutation library and primer sequences for constructing a 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, the DNA template is mixed with the NNK primer, amplified through the PCR program, and then the original template is degraded by DpnI enzyme, and then the PCR product is concentrated and purified by sodium acetate ethanol precipitation. The specific steps are as follows:

[0091] 1) PCR amplification: Prepare the PCR reaction system on ice, add 1 μL DNA template, 2.5 μL forward primer (10 μM), 2.5 μL reverse primer (10 μM) and 25 μL 2×PCR high-fidelity enzyme premix, add ddH2O to a total system of 50 μL, and perform cyclic amplification according to the procedure of pre-denaturation-denaturation-renaturation-extension-final extension, see Table 2.

[0092] Table 2 Site-directed mutagenesis PCR amplification program

[0093]

[0094] 2) Template degradation: After PCR amplification, place the PCR product on ice for 2 minutes to cool it down, then add 2μL DpnI enzyme, 6μL 10× Cutsmart buffer, and 2μL ddH2O to form a 60μL enzyme digestion system with the PCR product. Place at 37°C for 4-8h.

[0095] 3) Product purification:

[0096] ① Add 6 μL of sodium acetate (3M, pH 5.2) to the DNA solution and mix thoroughly;

[0097] ② Add 130 μL of ice-cold ethanol, mix thoroughly, and place in a –20°C refrigerator for 30 minutes;

[0098] ③ Centrifuge at 12000g for 5 minutes, carefully remove the supernatant, and aspirate all the droplets on the tube wall;

[0099] ④ Add 750 μL 70% ethanol, centrifuge at 12000 g for 2 minutes, carefully remove the supernatant, and aspirate all the droplets on the tube wall;

[0100] ⑤ Place the opened EP tube on a clean bench at room temperature for 8-10 minutes until there is no ethanol smell.

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

[0102] (3) Electric shock transformation

[0103] Take 2μL library DNA and 40μL DH5α electroporation competent cells and mix them thoroughly, carefully transfer them to the pre-cooled electroporation cup (be careful not to produce bubbles), and place them on ice for use; set the electroporation transformation parameters to 25μF, 200Ω, 1.8KV, and perform electroporation transformation. After the electroporation is completed, add 1mL SOC liquid culture medium to the tank, blow and mix, transfer the bacterial solution to a 10mL EP tube, add 1mL SOC liquid culture medium, mix, and place in a constant temperature shaking incubator at 37℃, 250rpm for 1h to recover. Spread all 2mL of bacterial solution evenly on a 25cm LB agar culture dish and culture it in a constant temperature incubator at 37℃ for 12 to 16h.

[0104] (4) Library extraction

[0105] Add 8-10 mL of LB liquid medium to the culture dish full of colonies, gently scrape the colonies with a sterile scraper and transfer them to a 50 mL centrifuge tube. Centrifuge at 10,000 rpm for 3 minutes, discard the supernatant, extract the bacteria according to the steps of the plasmid extraction kit, obtain the mutant library and store it in a -20°C refrigerator for future use.

[0106] (5) Sequencing verification

[0107] Before library extraction, a portion of monoclones were selected for culture in LB liquid medium, and then the plasmids were extracted and sent to the Xi'an Sequencing Department of Sangon Biotech Co., Ltd. for sequencing. The sequencing results need to have a certain diversity to ensure the integrity of the library.

[0108] The library capacity of this example is measured by the number of clones grown after the unit mass of the library plasmid is transformed, i.e., colony-forming units (CFU). The transformed bacterial solution is diluted by gradient dilution method, spread on a 10 cm culture dish, and counted after the colonies grow. For example, when the 1 ng / μL SMTT library is transformed and diluted 100 times, the number of colonies is 45, and the library capacity = 45×100CFU / 1ng = 4.5×10 6 CFU / μg.

[0109] The diversity of the library in this embodiment is evaluated by sequencing some library colonies and the ratio of the number of mutants to the total number of tests. In theory, the mutant type also includes the wild type wt, which we ignore in the experiment. The experimental results show that the proportion of mutants in the T1145 library and the T1146 library is less than 70%, indicating that the quality of these two libraries is poor, and the proportion of mutants in 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 mutation library based on BACTH system screening

[0111] The above saturation mutation library was screened by the BACTH system, and many mutants obtained by sequencing were still wild-type, so it was suspected that this situation might be caused by incomplete template degradation by the DpnI enzyme. In order to avoid interference from the wild type, we used the forward saturation mutation library to screen the inactive mutants of each mutation site through the BACTH system, and then used the inactive mutants as templates to construct the reverse saturation mutation library through the NNK primer to eliminate interference from the wild type.

[0112] (1) Obtaining inactive mutants

[0113] The mutation library constructed with T18-Hc / A as template is called forward saturation mutation library. 1μL forward saturation mutation library (1ng / μL) was mixed with 1μL T25-SV2C-L4 (10ng / μL), 100μL BTH101 chemical competent state was added, mixed, and placed in ice bath for 30min; heat shock in 42℃ metal bath for 90s, and ice bath again for 2min; 200μL LB liquid culture medium was added, and placed in a constant temperature shaking incubator at 37℃ and 250rpm for 45min for recovery. The bacterial solution was diluted a certain multiple and evenly spread on LB / X-Gal screening medium. After culturing in a 30℃ constant temperature incubator for 48h, 3 white monoclones were picked from each library and inoculated into a shaking bacterial tube containing 4mL LB liquid culture medium, and placed in a constant temperature shaking incubator at 37℃ and 250rpm for overnight culture. The plasmid was extracted according to the plasmid extraction kit the next day. The extracted plasmid was sent to the Xi'an Sequencing Department of Sangon Biotech Co., Ltd. for sequencing. If there was a mutation at the mutation site in the library, the sequencing was considered 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 chemical competence again by heat shock transformation. The transformation product is evenly spread on LB agar medium containing 100μg / mL Amp. T25-SV2C-L4 has Kan resistance but no Amp resistance, so it cannot grow in LB agar medium containing Amp. After culturing in a 37℃ constant temperature incubator for 12-16h, a single clone is picked and inoculated into LB liquid medium, and cultured in a constant temperature shaking incubator at 37℃ and 250rpm for 12-16h, and then the plasmid is extracted according to the plasmid mini-extraction kit, and the extracted plasmid is stored in a -20℃ refrigerator.

[0115] After plasmid purification, the T18-mutant and T25-SV2C-L4 were co-transformed into the chemically competent BTH101 again. After culturing in a 30°C constant temperature incubator for 48 h, if all colonies were white, it proved that the mutant was inactive.

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

[0117] The construction method was consistent with the forward saturation mutation library, the only difference was that the PCR template used an inactive mutant plasmid extracted from the white colonies in the forward mutation library.

[0118] The reverse saturation mutation library corresponding to the forward saturation mutation library was constructed using inactive mutants as templates to exclude the screening bias caused by wt residues. The library quality was evaluated by library capacity and mutant ratio. The results showed that the reverse library had 100% mutant ratio in the library because it excluded the disadvantages of using wt as a template for library construction. From the sequencing results of small-scale testing, all libraries tested were mutants, and the types of various mutants were different, indicating that the reverse library had good diversity and uniformity.

[0119] 1.3.3 Protein interaction analysis based on BACTH system

[0120] The forward saturation mutation library and the reverse saturation mutation library were screened multiple times by the BACTH system. After the blue colonies were picked for preliminary β-galactosidase activity determination, the plasmids with activity higher than wt were selected for sequencing to obtain the main mutation types at each site. Combining the single-site saturation mutation library and the combinatorial mutation library, it was found that S1142, M1144 and T1146 were active plastic areas. There were many types of amino acid substitutions at the S1142 site, mainly I / V / Q / F; the amino acid substitutions at the M1144 site were concentrated, mainly L / I / V; T1146 was replaced by two amino acids, T / S. T1145, Y1149, R1156 and R1294 were mainly wild-type, and Y1149F and R1156W / S appeared less frequently (as shown in Table 3).

[0121] The mutants S1142I / V / Q / F, M1144L / I / V, T1146S, Y1149F, and R1156S / W identified in the BACTH test were further analyzed for β-galactosidase activity. The results showed that mutants S1142I / V / Q / F, M1144L / I / V, and T1146 all exhibited strong β-galactosidase activity, indicating that these mutants have enhanced binding to the receptor SV2C ( Figure 1 shown).

[0122] Table 3 Saturation mutation library screening results

[0123]

[0124]

[0125] Note: The letters in LLTS etc. respectively represent the amino acid substitution types at S1142, M1144, T1145 and T1146 positions, wt represents the wild type, and “-” means no active mutants were screened out.

[0126] Based on the screening results of the forward saturation mutation library and the reverse saturation mutation 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 mutations per pair, and combined with T1146S, there will be 24 mutants. We screened a combination of mutant libraries to obtain a portion of mutants: FLTT (S1142F / M1144L), VLTS (S1142V / M1144L / T1146S), IVTT (S1142I / M1144V), VVTT (S1142V / M1144V), QVTT (S1142Q / M1144V), FITT (S1142F / M1144I), and HWTT (S1142H / M1144W). Based on this, we constructed ILTT (S1142I / M1144L), VLTT (S1142V / M1144L), QLTT (S1142Q / M1144L), IVTT (S1142I / M1144V), and HWTT (S1142H / M1144W). The combined mutants of S1142F / M1144V (S1142H / M1144W / T1146S), S1142H / M1144W / T1146S), S1142F / M1144L / T1146S), S1142F / M1144V / T1146S), S1142F / M1144V / T1146S), S1142I / M1144I / T1146S), S1142I / M1144I / T1146S), S1142I / M1144I / T1146S), S1142Q / M1144I / T1146S), S1142I / M1144I / T1146S), S1142I / M1144I / T1146S), S1142I / M1144V / T1146S, and S1142F / M1144V / T1146S were successfully constructed.

[0127] The β-galactosidase activity of various combined mutants was compared with that of wt, and all of them had a significant improvement ( Figure 2 Among them, the activities of mutants such as VVTT, VLTT, VLTS, FLTT, FLTS, and HWTT were relatively high, so these mutants were selected for subsequent purification and used for the next 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 the Nde I and EcoR I sites of the pET28a(+) vector to obtain the pET 28a(+)-Hc / A1 recombinant plasmid for the preparation of His6-Hc / A1 wild-type and mutant proteins. The mutant recombinant plasmid was prepared using the Yisheng Biotechnology Multi-site Directed Mutagenesis Kit (Cat. No. 11004ES10) according to the manufacturer's instructions.

[0131] The SV2C-L4 sequence was synthesized by GeneWei Corporation and cloned into the BamHI and EcoRI sites of the 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 chemical competent cells were from Sangon Biotech Co., Ltd.

[0133] The pET 28a(+)-Hc / A1 wt and mu mutant recombinant vectors successfully constructed by sequencing were transformed into BL21(DE3) competent cells by conventional heat shock transformation method, and the pGEX-4T-1-SV2C-L4 recombinant vector was transformed into DH5α competent cells by conventional heat shock transformation method. Single clones were picked one by one and inoculated into 20mL LB medium containing 80μg / mL Kan, and pGEX-4T-1-SV2C-L4 was inoculated into LB medium containing 100μg / mL Amp, and the culture was shaken at 37℃, 200rpm overnight to prepare seeds. Take the above bacterial solution, inoculate 5% into the above 500mL LB medium containing appropriate antibiotics, and culture at 37℃, 200rpm until OD 600 When the pH was 0.6, IPTG was added at a final concentration of 0.2 mM to induce expression for 5 hours, and the bacteria were collected by centrifugation at 8000 rpm for 5 min.

[0134] Take the above-mentioned bacteria and add 20mM PB pH7.4 buffer at a ratio of 1:30, ultrasonically disrupt the bacteria until the solution is clear, centrifuge at 10000rpm for 10min, and take the supernatant. His6-Hc / A1 wt and each mutant were purified by affinity chromatography using Cytiva's Ni Sepharose 6FF filler, and GST-SV2C-L4 was purified by affinity chromatography using Cytiva's Glutathione Sepharose 4FF filler. The purification operation was carried out according to the instructions of the filler manufacturer. The purified His6-Hc / A1 wt and each mutant and GST-SV2C-L4 were dialyzed against 20mM PB pH7.4 overnight, and the target protein was obtained to determine the protein concentration by the BCA method. Among them, His6-Hc / A1 wt and each mutant, except VLTS at 7 μg / mL, the others were all 50 μg / mL, and GST-SV2C-L4 was 100 μg / ml. The above protein solutions were divided into small portions and frozen at -20°C.

[0135] 2.2 Reagents

[0136] Series S Sensor Chip CM5 (GE, USA, Cat. No. 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 HBS-EP+buffer and deionized water on the corresponding brackets, and connect the corresponding pipes. After the instrument runs stably, open the chip door, 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 fixing the ligand on the CM5 chip, it is necessary to screen the appropriate ligand buffer pH so that the ligand can be enriched near the chip surface through electrostatic adsorption to achieve a better coupling effect. Usually, it is screened from Acetate 4.0, Acetate 4.5, and Acetate 5.0; GST-SV2C-L4 is diluted to 5 ng / μL with Acetate 4.0, Acetate 4.5, and Acetate 5.0, respectively, with a binding time of 180 s and a flow rate of 10 μL / min, and then the chip surface is regenerated with 50 mM NaOH.

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

[0146] 0.4M EDC and 0.1M NHS in the amino coupling kit were mixed in a ratio of 1:1, and flowed through the chip surface Fc2 at a flow rate of 10μL / min for 200s to activate the carboxymethyl dextran matrix on the chip surface; GST-SV2C-L4 was diluted to 5ng / μL with Acetate4.0 and flowed through the activated chip surface at a flow rate of 10μL / min to covalently couple GST-SV2C-L4 to the chip surface, and the target coupling amount was set to 200RU; the activation sites of uncoupled proteins on the chip surface were blocked with ethanolamine for 200s.

[0147] The same operation was used to activate and block Fc1 on the chip surface as a reference channel.

[0148] (4) Kinetic analysis

[0149] Hc / Awt and S1142H / M1144W (HWTT) were diluted to 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125μM, and 7.8125nM respectively with HBS-EP+buffer as repeated concentrations to test the repeatability of the experiment; S1142V / M1144L (VLTT), S1142V / M1144L / T1146S (VLTS), S1142F / M1144L (FLTT), S1142F / M1144L / T1146S (FLTS), and S1142V / M1144V (VVTT) were diluted 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, respectively, as repeated concentrations to test the repeatability of the experiment.

[0150] Different concentrations of protein ligands flowed through 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 Glycine 2.0, and the regeneration time was 30 s; after the kinetic program was completed, the kinetic results were fitted using Biacore T200 analysis software. The kinetic parameters of S1142F / M1144L, S1142V / M1144L / T1146S binding to SV2C-L4 are shown in Table 4.

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

[0152] Hc / A <![CDATA[k on (1 / Ms)]]> <![CDATA[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 value in Table 4 indicates the affinity of the mutant to the receptor SV2C. It can be seen that the affinity of the Hc / A mutants screened and obtained by the present invention to the extracellular domain of the receptor is ranked as follows: FLTT>VLTS>VLTT>FLTS>VVTT>HWTT>wt. Among them, FLTT has the strongest affinity to the receptor SV2C, which is 6.3 times that of WT to the receptor SV2C; followed by VLTS, which is 5.6 times that of wt.

[0154] Example 3 Detection of the enzymatic activity of BoNT / A mutants on substrate SNAP-25 at the cell level

[0155] 3.1 Biomaterials

[0156] BoNT / A1 wt, 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 Yisheng Biotechnology. The SDS-PAGE results of the purified target protein are shown in Figure 3 ; SD pregnant mice (SPF grade) at 18 days of gestation were from Lanzhou Veterinary Research Institute, license number SCXK (Gan) 2020-0002.

[0157] 3.2 Reagents

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

[0159] β-actin antibody (Immunoway, cat. no. YM3028);

[0160] RIPA lysis buffer (Solyb, Cat. No. R0010);

[0161] NeurobasalTM Medium (Thermo Fisher Scientific, Cat. No. 21103-049);

[0162] B-27Supplement (Thermo Fisher Scientific, Cat. No. 17504-044);

[0163] GlutaMAXTM-1 (Thermo Fisher Scientific, Cat. No. 35050-061).

[0164] 3.3 Implementation steps

[0165] (1) Extraction of hippocampal neurons

[0166] Preliminary preparation: Poly-lysine coated coverslips, placed coverslips that have been washed with 0.2M HCl and disinfected with 75% ethanol in a 24-well plate, added 0.1mg / mL poly-lysine, 100μL-200μL per well, and placed at room temperature overnight. Aspirate the poly-lysine, wash with sterile water 3 times, and blow dry for later use.

[0167] Isolate the hippocampus: kill the pregnant mouse with high concentration of CO2, remove the embryo, cut off the head of the fetus, and put it in a culture dish containing dissection fluid; transfer to the clean bench, observe and separate the hippocampus under a dissecting microscope, put it in a centrifuge tube containing dissection fluid, and place it in an ice box; aspirate the dissection fluid, add 0.05% preheated trypsin, and digest it in a 37°C water bath, shaking every 10 minutes; aspirate the trypsin, add 10mL of precooled dissection fluid each time, flip the centrifuge tube 5 times, and wash 3 times; aspirate the dissection fluid, add 1mL of neuronal culture medium, press against the bottom of the centrifuge tube, blow 8 times, disperse the block tissue into single cells, and avoid generating bubbles; add the corresponding volume of neuronal culture medium, flip the centrifuge tube to disperse the cells evenly, 600μL per well, and inoculate it in a 24-well plate coated with polylysine; place it in a 37°C constant temperature incubator and culture it for 12 days for subsequent experiments.

[0168] (2) Western Blot

[0169] BoNT / Awt and mutant FLTT and VLTS were diluted with neuron culture medium to 7.5pM, 15pM, 31.25pM, 62.5pM, and 125pM series gradient concentrations. The corresponding concentrations of botulinum toxin were added to the neurons in the 24-well plate, mixed and incubated in a 37°C constant temperature incubator for 48h. 70μL RIPA lysis buffer was added to each well of cells and lysed at 4°C for 10min. Then centrifuged at 4°C and 12,000g for 10min, 5×SDS-PAGE protein loading buffer was added to the supernatant, and denatured at 100°C for 10min. The SDS-PAGE electrophoresis conditions were: 80V pressing glue for 30min, then 120V for 1.5h. Cut the PVDF membrane of appropriate size, activate it in methanol for 20s, and stick the PVDF membrane and glue together according to the structure of cellulose pad-filter paper-PVDF membrane-glue-filter paper-cellulose pad, 300mA, and transfer the membrane for 2h. After transfer, use TBST buffer to prepare 5% skim milk powder solution as blocking solution, and block at room temperature for 2 hours. Dilute the primary antibody at 1:5,000 and incubate at 4°C overnight. Wash the membrane 3 times with TBST buffer, 60rpm, 10min / time. Dilute the secondary antibody at 1:10,000 and incubate at room temperature for 1 hour. Wash the membrane 3 times with TBST buffer, 60rpm, 10min / time. Use ECL luminescent solution to develop protein bands, record with gel imaging system, and take pictures.

[0170] The results of BoNT / A1 wt, FLTT and VLTS are as follows Figure 4 shown. Figure 4 The results in a show that mutants FLTT and VLTS showed obvious SNAP-25 cleavage at a low concentration of 7.5pM, while BoNT / A1 wt showed the ability to cleave SNAP-25 at 15pM. The lower the toxin concentration corresponding to the cleavage of SNAP-25, the stronger the toxin effect. Therefore, mutants FLTT and VLTS showed stronger SNAP-25 cleavage efficiency than wt. The gray value of the Western Blot results was measured, and a line graph of the percentage of cleaved SNAP-25 and toxin concentration was drawn, which further intuitively reflected that the ability of mutants FLTT and VLTS to substrate SNAP-25 was better than that of BoNT / A1 wt ( Figure 4 b and c).

[0171] Example 4 Study on the activity, lethality and spread of BoNT / A mutants at the animal level

[0172] 4.1 Biomaterials

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

[0174] Clean-grade Kunming mice were purchased from the Experimental Animal Center of Lanzhou University with license number SCXK (Gansu) 2023-0003.

[0175] 4.2 Implementation steps

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

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

[0178] 4.2.2 Functional study of BoNT / Awt and its mutants based on mouse DAS scores

[0179] The toe abduction score (DAS) is a physiological model for assessing muscle relaxation. The model is based on the fact that mice, when briefly suspended by their tails, elicit a typical startle response characterized by hind limb extension and toe abduction. The model is based on a 5-point scale (0-4 points), 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 assay, the LD values ​​of wt and its mutants FLTT and VLTS were calculated. 50 , on this basis, set the gradient concentration, set each injection of 60pg, 30pg, 15pg, 5pg, 1pg, 0.5pg six dose groups, injection volume of 10μL. The mice were randomly divided into 3 groups, each group was randomly divided into 6 dose groups, and each mouse was weighed and marked one day before injection. The gastrocnemius muscle of the left hind limb of the mouse was injected intramuscularly. Within the first 12h of the injection, the DAS score was performed every 2h, and then once every 12h or once a day, and the weight of the mouse was recorded daily.

[0181] 4.2.3 Mouse Fluorescence Imaging Experiment

[0182] Hc / Awt and its heavy chain receptor-binding domain mutants FLTT and VLTS were purified using Ni 2+ affinity chromatography. Then, the buffer was exchanged by dialysis with 0.1 M NaHCO3. After that, 10 μL of SF680 fluorescent dye was added per milliliter of protein and incubated at room temperature for 3 hours. Finally, the buffer was exchanged by dialysis with PBS buffer. After shaving the back hair of the mice, 2 μL of Hc / Awt and mutants Hc / AFLTT and VLTS were injected into the back muscles. Then, fluorescence imaging was performed using a small animal in vivo imaging system at 0 hours and 8 hours respectively. The fluorescence area was calculated and statistically analyzed using Image J software.

[0183] 4.2 Implementation Results

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

[0185] Table 5 Detection Results Related to the Efficacy, Safety, and Diffusivity of BoNT / A Mutant Toxins in Vivo

[0186] BoNT / A1 <![CDATA[ED 50 (pg)]]> 0%BW(pg) <![CDATA[0%BW / ED 50 ]]> <![CDATA[ip LD 50 (pg)]]> <![CDATA[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 median lethal dose by intraperitoneal injection ip LD 50 value 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 calculation results: wt < FLTT = VLTS, indicating that the lethal activities of mutants FLTT and VLTS are 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 animal death. The median lethal dose by intramuscular injection im LD 50 can to a certain extent reflect the diffusivity of the toxin. Therefore, the larger the value, the lower the diffusivity of the toxin. According to the im LD 50 results: VLTS > FLTT > wt, suggesting that the diffusivities of mutants VLTS and FLTT are significantly lower than that of wt (Table 5).

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

[0189] A 0% body weight change (%BW) represents the toxin dose that does not cause any reduction in body weight, thereby reflecting the extent 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 diffusibility compared to wt( Figure 6 ).

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

[0191] In the DAS scoring experiment, the 0% BW value of the mice and the im LD 50 value analysis in the mouse bio - lethality determination experiment showed that the diffusibility of mutants FLTT and VLTS is lower than that of wt. To further measure the diffusion range of intramuscular injection of the toxin, we purified Hc / Awt, Hc / AFLTT, and VLTS, and used a small animal in - vivo imaging system to actually measure the diffusion area after a period of time of toxin injection. It was found that the actually measured fluorescence area of VLTS (0.394 ± 0.045 cm 2 ) and the actually measured fluorescence area of FLTT (0.470 ± 0.045 cm 2 ) are significantly lower than the actually measured fluorescence area of wt (0.561 ± 0.039 cm 2 ), indicating that the diffusibility of mutants FLTT and VLTS is lower than that of wt( Figure 7 ).

[0192] The mutants BoNT / A - FLTT and BoNT / A - VLTS prepared in the present invention have lower diffusibility, higher safety, and improved ED 50 compared to the wild - type. Defining ED 50 as 1 U, 25 U of BoNT / Awt, BoNT / A - FLTT, and BoNT / A - VLTS were respectively injected into the gastrocnemius muscle of the left hind limb of mice. The results showed that at 5 days after injection, all the mice in the BoNT / A wt group died, 62.5% of the mice in the BoNT / A - FLTT group survived, and all the mice in the BoNT / A - VLTS group survived. Figure 8 ).

[0193] The main drawbacks of the clinical application of BoNT / A1 products are that the diffusion generated at the injection site will cause side effects to the human body, and some people will produce neutralizing antibodies, which will make the treatment effect worse or ineffective. The present invention modifies the BoNT / A1 receptor binding domain Hc / A1 in order to improve the binding ability of Hc / A1 with the receptor SV2C (SV2C-L4 is the extracellular domain of SV2C), increase the ability of the toxin heavy chain HC to mediate its light chain LC to enter the target cell, reduce the diffusibility of the toxin, thereby improving its efficacy, achieving the purpose of reducing the amount of toxin used, and then reducing the production of neutralizing antibodies.

[0194] The present invention uses the BACTH system to perform saturation mutation screening on the binding domain-related sites of Hc / A1 and SV2C-L4, and determines the in vitro affinity constants of some of the screened mutants. The obtained Hc / A1 mutants are recombined into the full-length BoNT / A1 toxin to evaluate the effect of Hc / A1 mutation modification on the activity of BoNT / A1 at the hippocampal neuron cell level and the whole mouse animal level.

[0195] The present invention uses the BACTH system to perform saturation mutation screening on the key amino acid residues of the binding domain of Hc / A1 and SV2C-L4, and determines the dominant amino acids and modifiable sites of these sites. Among them, S1142, M1144 and T1146 are active plastic regions, and suitable substitutions may improve the affinity of HC / A and receptor SV2C. When S1142 is substituted with I / V / Q / F, the activity is higher, and the amino acid changes at M1144 are more concentrated, mainly substituted with the three amino acids I / L / V or W. The main similar substitution at T1146 is S, but it may cause loss of activity in individual mutants. T1145, Y1149, R1156 and R1294 are active non-plastic regions, and it is not recommended to perform mutation screening on these four sites of Hc / A1.

[0196] The present invention further performs β-galactosidase activity determination and SPR affinity detection on several mutants with better activity obtained by screening, and determines two mutation combinations with improved activity compared with wt, among which the affinity of FLTT to SV2C is increased by about 6.3 times compared with wt, and the affinity of VLTS to SV2C is increased by about 5.6 times compared with wt.

[0197] The present invention is based on the detection of SNAP-25 cleavage activity during the whole intoxication process of botulinum toxin at the cellular level. The cleavage ability of BoNT / A1 full-length mutants FLTT and VLTS for SNAP-25 is significantly higher than that of BoNT / A1 wt.

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

[0199] Starting from the amino acid sequence of the BoNT / A1 mutant disclosed in the present invention, its nucleotide sequence can be inferred using a codon table, and after full synthesis using a codon optimization method, the BoNT / A1 mutant gene sequence can be made more suitable for expression in different host cells without affecting the primary amino acid structure of the protein of the expression product, which can be understood by those skilled in the art.

[0200] The present invention discloses that the S1142, M1144, and T1146 of the mutated BoNT / A1 heavy chain receptor binding domain and its receptor binding interface are replaced or not replaced with FLT or VLS. Accordingly, the heavy chains of other subtypes or other toxin serotypes and their subtypes that use SV2 as a receptor are subjected to the same mutations at the sites homologous to the amino acid mutation sites in the present invention by sequence alignment, thereby reducing the diffusivity of the toxin and improving its safety, which can be understood by those skilled in the art.

[0201] BoNT / A1 can treat a wide range of diseases clinically. BoNT / A1 was first used in ophthalmology to treat strabismus in the late 1970s. Its current indications mainly include blepharospasm, spastic dystonia, chronic migraine, etc. In dermatology, botulinum toxin is used to temporarily improve moderate to severe frown lines, remove wrinkles, and slim the face and legs, treat hyperhidrosis, and prevent surgical scars. In recent years, the application potential of BoNT / A1 in the fields of depression and tumor treatment has gradually emerged. In addition, it can also be used in neurology: spasmodic torticollis, hemifacial spasm, etc.; nerve injury rehabilitation treatment: spinal cord injury, neurogenic bladder, and brain trauma; acting on parasympathetic synapses: excessive secretion of sweat glands (hyperhidrosis, excessive tearing), body odor; muscle spasms caused by the latter, etc.; inhibiting the release of peptides in the peripheral trigeminovascular system: treating migraine and toothache; others: cardia spasm, various sphincter spasms, constipation, etc. BoNT / A1 product Botox appeared on the cover of Time magazine on January 16, 2017, claiming that it can also be used to treat 793 other diseases.

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

[0203] In summary, the beneficial effect of the present invention is that after mutation modification of Hc / A of BoNT / A, its affinity with the receptor SV2C is improved. By replacing the wild-type Hc / A in the full-length toxin, its activity of cleaving the substrate SNAP-25 and its ability to cause muscle paralysis are improved, its diffusivity is reduced, and its safety is improved. The botulinum toxin type A mutant disclosed in the present invention is added with pharmaceutically acceptable buffer components, salts and other auxiliary ingredients to form a pharmaceutical composition, which can be used in the preparation of drugs for treating related diseases that can be treated by wild-type toxins or related medical beauty products. At the same time, BoNT / A mutants can be used as drug carriers for targeted delivery of drugs to the nervous system.

Claims

1. A botulinum toxin type A mutant, characterized in that: Compared to wild-type botulinum toxin type A BoNT / A1, the mutant is selected from: (1) any one of the mutated amino acids S1142F, S1142V, S1142Q, S1142I, and S1142H; and / or, (2) any one of the amino acid mutations among M1144L, M1144V, M1144W, and M1144I; and / or, (3) Amino acid of T1146S mutation.

2. The botulinum toxin type A mutant according to claim 1, characterized in that The mutant is selected from any one of the mutated amino acids S1142I, S1142V, S1142Q, S1142F, M1144L, M1144I, M1144V, and T1146S.

3. The botulinum toxin type A mutant according to claim 1, characterized in that The mutant is selected from the mutated amino acids described in any one of the following groups: S1142F and M1144I; S1142I and M1144V; S1142V and M1144V; S1142Q and M1144V; S1142F and M1144V; S1142I and M1144L; S1142V and M1144L; S1142Q and M1144L; S1142F and M1144L; S1142H and M1144W.

4. The botulinum toxin type A mutant according to claim 3, characterized in that: The mutant is selected from the mutated amino acids described in any one of the following groups: S1142V and M1144V; S1142V and M1144L; S1142F and M1144L; S1142H and M1144W.

5. The botulinum toxin type A mutant according to claim 4, characterized in that The mutant is an amino acid with mutations of S1142F and M1144L; the amino acid sequence of the mutant is shown in SEQ ID NO.

2.

6. The botulinum toxin type A mutant according to claim 1, wherein The mutant is selected from the mutated amino acids described in any one of the following groups: S1142V, M1144I and T1146S; S1142Q, M1144I and T1146S; S1142V, M1144V and T1146S; S1142V, M1144L and T1146S; S1142F, M1144L and T1146T; S1142H, M1144W and T1146S.

7. The botulinum toxin type A mutant according to claim 6, characterized in that The mutant is selected from the mutated amino acids described in any one of the following groups: S1142V, M1144L and T1146S; S1142F, M1144L and T1146T.

8. The botulinum toxin type A mutant according to claim 7, characterized in that The mutants are amino acids with mutations of S1142V, M1144L and T1146S; the amino acid sequence of the mutants is shown in SEQ ID NO.

3.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the botulinum toxin type A mutant according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. Use of the botulinum toxin type A mutant according to any one of claims 1 to 8 or the composition according to claim 9 in the preparation of therapeutic drugs, medical cosmetic products, or drug targeted delivery vectors; The therapeutic drug comprises at least one of improving / treating blepharospasm, strabismus, spastic dystonia, migraine, and tumor; The medical cosmetic products include at least one of those for improving moderate to severe frown lines, removing wrinkles, slimming the face, and slimming the legs; The drug targeting delivery vector includes a delivery vector for drugs targeting the nervous system or SV2C receptors.

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