Botulinum neurotoxin variants, constructs, compositions, and methods of use

Abstract

This document discloses clostridial neurotoxin (BoNT) or SNARE cleavage homolog variants, constructs, and methods of use thereof. The BoNT or SNARE cleavage homolog variants may include substitutions, deletions, or insertions of one or more amino acids or domains in the light chain.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 584,393, filed September 21, 2023, and U.S. Provisional Application No. 63 / 617,936, filed January 5, 2024, each of which is incorporated herein by reference in its entirety.

[0003] Statement regarding federally funded research or development

[0004] This invention was completed with government support under license number R01AI139306 granted by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0005] Reference to electronic sequence listing

[0006] The contents of the electronic sequence list (65005301014.xml; size: 134,395 bytes; creation date: September 22, 2024) are included in this paper by reference in full. Background Technology

[0007] Clostridium botulinum is a Gram-positive anaerobic bacterium that produces botulinum neurotoxins (BoNTs), the most potent proteins in humans. BoNTs are 150 kDa zinc-dependent metalloproteinases with a 50-kDa N-terminal catalytic light chain (LC) and a 100 kDa C-terminal heavy chain (HC). The HC contains an LC translocation domain (HCN) and a receptor-binding domain (HCC), enabling binding to neurons and subsequent endocytosis and translocation of the LC to the cytoplasm. BoNTs comprise seven pathogenic serotypes (AG); BoNT / A comprises eight subtypes (BoNT / A(1-8)), which specifically cleave the 25 kDa synaptosome-associated protein (SNAP-25) on the cytoplasmic side of the neuronal plasma membrane. Summary of the Invention

[0008] In one aspect, a method is provided. The method may include administering a synthetic mRNA construct to a subject in need to produce a clostridial neurotoxin (BoNT) or a SNARE-cleaving homologous light chain or a variant thereof.

[0009] In another aspect, synthetic mRNA constructs for generating clostridial neurotoxin (BoNT) or SNARE-cleaving homologous light chains or variants thereof are provided.

[0010] In yet another aspect, a method for treating a subject is provided. The method may include administering a therapeutically effective amount of one or more synthetic mRNA constructs disclosed herein.

[0011] In another aspect, synthetic mRNA constructs for generating clostridium neurotoxin (BoNT) light chain (LC) or variants thereof are provided.

[0012] In yet another aspect, a therapeutic composition is provided comprising one or more synthetic mRNA constructs disclosed herein.

[0013] In another aspect, a therapeutic composition is provided. The therapeutic composition may comprise a clostridium neurotoxin (BoNT) variant, wherein the BoNT variant includes N-terminal, membrane localization domain (MLD), or both domain substitutions.

[0014] In yet another aspect, a method for treating a subject is provided. The method may include administering a therapeutically effective amount of one or more mRNA constructs disclosed herein and / or one or more therapeutic compositions disclosed herein. Attached Figure Description

[0015] Figure 1 Cytoskeleton inhibitors revealed the transport of LC / A1 along microtubules. Following overnight transfection, N2A cells were treated for 60 min with a culture medium control, 4 µM cytochalasin D, or 9 µM nocodazole, and then fixed with 4% paraformaldehyde. N2A cells were imaged using EGFP fluorescence (excitation wavelength 488 nm, emission wavelength 509 nm), phalloidin (excitation wavelength 555 nm, emission wavelength 605 nm), and β-tubulin (excitation wavelength 645 nm, emission wavelength 705 nm). Representative images show the homeostatic intracellular localization of EGFP, EGFP-LC / A1, EGFP-LC / A3V, and EGFP-LC / A3V.

[0016] Figure 2A and 2B LC / A1 co-localizes with Rab GTPase. Figure 2A Following overnight transfection and fixation with 4% paraformaldehyde, and staining with Rab3a, / 4, / 5, / 7, / 11, and / 27a, N2A cells were imaged using EGFP fluorescence (excitation wavelength 488 nm, emission wavelength 509 nm) and α-Rab (excitation wavelength 645 nm, emission wavelength 705 nm). Representative images show homeostatic intracellular localization and co-localization with RAB GTPases indicated by white dots. Figure 2B LC / A1 was plotted against the indicated Rab: 3a, 4, 5, 7, 11, or 27a, and plotted using Graphpad Prism 9.3.1. Results were obtained by averaging three replicates from three independent experiments.

[0017] Figure 3A and3B Dominant and negative Rab GTPases isolate LC / A1 into intracellular vesicles. Figure 3A After overnight co-transfection with EGFP-dominant / negative Rab protein and DsRed-A1 and fixation with 4% paraformaldehyde, N2A cells were imaged using EGFP-DNRab fluorescence (excitation wavelength 488 nm, emission wavelength 509 nm) and DsRed-A1 fluorescence (excitation wavelength 555 nm, emission wavelength 605 nm). Representative images show the imaging of EGFP-dominant / negative Rab protein and DsRED-A1, respectively, and their combined steady-state intracellular localization. Controls of EGFP-LC / A1 and DsRed-LC / A1 were analyzed to ensure that DsRed-LC / A1 did not alter the membrane localization of LC / A1. Figure 3B Percentage of cells containing membrane signals. Ten randomized fields of view were selected from three independent experiments, and membrane localization was counted. Means and SEM values ​​were assessed using GraphPad Prism 9.3.1 by ordinary one-way ANOVA, with Dunnett's multiple comparison test using DsRed-A1 as the control column. ns - not significant; all other values ​​are not significant. p<0.0001. The result is the average of three replicates from three independent experiments.

[0018] Figure 4Transport of botulinum neurotoxin LC / A1 after translocation in Neuro-2A cells. (1) BoNT / A1 binds to gangliosides and synaptic vesicle protein-2 (SV2) on the surface of motor neurons. (2) BoNT / A1 is endocytosed in early Rab5 endosomes (3) and then transported to sorting endosomes, where (4a) BoNT / A1 is transported into Rab4 for rapid endosome recovery. After acidification, LC / A1 translocates through pores formed by HCC. (4b) BoNT / A1 is transported into Rab11 for endosome recovery, which promotes slow recovery because DN Rab11 does not inhibit LC / A1 membrane localization; this pathway is secondary to Rab4 recovery. (4c) A portion of BoNT / A1 is transported into late Rab7 endosomes and retrogradely transported toward the nucleus. (5) Following translocation, LC / A1 binds to Rab3 and Rab27 synaptic vesicles (SVs) facilitated by the N-terminus (N) (red, residues 1-17) for (6) anterograde transport along membrane-associated microtubules (7), where the low homology domain (LHD) (green, A1 residues 275-357) localizes and cleaves LC / A1 via recognition of SNAP-25. EE, early endosome; HCN, translocation domain; HCC, receptor-binding domain; LC, light chain; LE, late endosome; 3, Rab 3; 4, Rab 4; 5, Rab 5; and 27, Rab 27; RE, recycle endosome; SE, sort endosome; SV2, synaptic vesicle protein-2.

[0019] Figure 5 Determining the boundaries of experimental Pearson colocalization coefficients. (Left) N2A cells were transfected with EGFP-LC / A1, then incubated with α-EGFP antibody and stained with DAPI for DNA. A representative deconvolution epifluorescence image is shown. Scale bar is 5 μm. (Right) Pearson colocalization coefficient plotted using GraphPad Prism 9.3.1.

[0020] Figure 6 EGFP-TRAP co-precipitation of the EGFP-LC construct. Performed according to the protocol described above. For transfection with the EGFP-LC construct encoding the indicated EGFP-TRAP fusion protein (… Cell lysates of N2A cells containing DNA were subjected to EGFP-TRAP immunoprecipitation. Samples were run on 13.5% SDS-PAGE, transferred to a PVDF membrane, and EGFP, actin, and β-3-tubulin were detected using 1:2000 rat α-EGFP monoclonal IgG (1:20000 mouse α-β-actin monoclonal IgG or 1:2000 rabbit α-β-tubulin). The bound primary antibody was identified using 1:10,000 goat-α-rat, 1:20000 goat-α-mouse, or 1:10,000 goat-α-rabbit conjugated with horseradish peroxidase, and detected using SuperSignal as described in the Methods section. An EGFP signal of approximately 75 kDa was detected in the lysate expressing the EGFP-fusion protein, while the lysate expressing only EGFP produced an (EGFP) reaction band at approximately 26 kDa, an actin reaction band at approximately 42 kDa, and a β-3 tubulin reaction band at approximately 50 kDa.

[0021] Figure 7 Dominant-negative Rab GTPases affect the endocytosis of Ctx-B and transferrin. (Left) N2A cells were treated with cholera toxin b subunit (Ctx-B) at 37°C for 5 min after overnight co-transfection with EGFP-dominant-negative Rab; cells were then fixed with 4% paraformaldehyde; N2A cells were imaged with EGFP fluorescence (excitation wavelength 488 nm, emission wavelength 509 nm) and Ctx-B (excitation wavelength 645 nm, emission wavelength 702 nm). Representative images show imaging of EGFP-dominant-negative Rab GTPases and Ctx-B, respectively, and their combined steady-state intracellular localization. (Right) After overnight co-transfection with EGFP-dominant-negative Rab GTPase, cells were treated with transferrin at 37°C for 5 minutes; subsequently, cells were fixed with 4% paraformaldehyde; N2A cells were imaged using EGFP fluorescence (excitation wavelength 488 nm, emission wavelength 509 nm) and transferrin (excitation wavelength 645 nm, emission wavelength 705 nm). Representative images show imaging of EGFP-dominant-negative Rab GTPase and transferrin, respectively, and their combined steady-state intracellular localization.

[0022] Figure 8Coomassie Brilliant Blue staining gel of purified rA3Q7P,V14G toxin: Purified recombinant BoNT / A3-Q7P,V14G was reduced (R) or unreduced (NR) by DTT and separated by SDS-PAGE gel chromatography, stained with InstaBlue (MidSci) total protein stain. A protein ladder (Seeblue2, Life Technologies) was run alongside the proteins for molecular weight determination of the protein bands. BoNT / A3-Q7P,V14G was almost completely reduced to 100 and 50 kDa heavy and light chain bands, indicating the formation of a proper double-stranded structure.

[0023] Figure 9 Duration of action in vivo for BoNT / A3 and BoNT / A3-Q7P,V14G. Indicative doses of BoNT / A3 or BoNT / A3-Q7P,V14G were injected into the gastrocnemius muscle of mice. Global motor neuron deficits were monitored by the rotarod test, and local paralysis was monitored by the DAS test. N=5.

[0024] Figure 10 Duration of action of BoNT / A3 and BoNT / A3-Q7P, V14G in primary neurons. Primary rat spinal cord cells were exposed to BoNT / A3 wt or BoNT / A3-Q7P, V14G, and cell samples were collected in triplicate at indicated time points. Cell lysates were analyzed by Western blotting and densitometry to determine the percentage of SNAP-25 cleaved by BoNT. The percentage of uncleaved SNAP-25 over time is shown, reflecting cellular recovery from BoNT poisoning.

[0025] Figure 11 BoNT LC activity in motor neurons transfected with mRNA. HiPSC-derived motor neurons were transfected with the indicated mRNA construct, and cell samples were collected at indicated time points (n=5). The mean and standard deviation of the percentage of SNAP-25 cut at each time point were determined, and the data points were fitted with a logarithmic trend line.

[0026] Figure 12 Human motor neurons were transfected with ciLNP-formulated LC / A1 mRNA. The duration of exposure of hiPSC-derived motor neurons to ciLNP-formulated LC / A1 mRNA was continuously indicated, and SNAP-25 cleavage of cell lysates was analyzed by Western blotting and densitometric analysis (n=2).

[0027] Figures 13A-13C . Figure 13AThe three-dimensional structure of the BoNT / A1 light chain was depicted. Figure 13B An enlarged view of the MLD structural domain portion is depicted. Figure 13C It involves sequence alignment of MLD regions in various BoNT serotypes. Figure 13C The sequence from top to bottom is SEQ ID NO: 56-66.

[0028] Figure 14 BoNT LC activity in motor neurons transfected with mRNA encapsulated in LNPs. HiPSC-derived motor neurons were transfected with the indicated mRNA construct, and cell samples were collected at indicated time points (n=5). The mean and standard deviation of the percentage of SNAP-25 cut at each time point were determined, and the data points were fitted with a logarithmic trend line.

[0029] Figures 15A-15CLC / A1 is transported along microtubules and co-localizes with Rab GTPase. (A) N2A cells were treated with cytochalasin D and nocodazole (control) or untreated after overnight transfection with pEGFP-LC / A1, pEGFP-LC / A3, or pEGFP (control) 60 minutes before fixation with 4% paraformaldehyde. N2A cells were imaged with EGFP fluorescence (green; excitation 488 nm, emission 509 nm), phalloidin (actin, Alexa568, red; excitation 555 nm, emission 605 nm), and β-tubulin (microtubules, primary antibody: rabbit α-β tubulin, followed by goat anti-rabbit-Alexa647 magenta; excitation 645 nm, emission 705 nm). Representative images show the homeostatic intracellular localization of EGFP (GFP), EGFP-LC / A1 (LC / A1), and EGFP-LC / A3LM (LC / A3) in untreated cells (control) or N2A cells treated with cytochalasin D or nocodaazole. Starting 7 hours post-pEFGP-LC / A1 transfection, N2A cells were treated with cytochalasin D for 60 minutes, followed by live-cell imaging every 15 seconds for 15 minutes. Bidirectional movement of EGFP-LC / A1 was detected in neuronal processes; data are not shown. (B) After overnight transfection with pEGFP-LC / A1, N2A cells were fixed with 4% paraformaldehyde, and individual Rab GTPases were probed (primary antibody: rabbit α-Rab IgG, followed by secondary antibody goat anti-rabbit-Alexa568) and EGFP-LC / A1 (EGFP: green) and individual Rab3a, Rab4, Rab5, Rab7, Rab11 or Rab27a (α-Rab: red). Representative images show the homeostatic intracellular localization of individual Rab GTPases (Rab), EGFPLC / A1 (GFP-LC / A1), merged images of individual Rab GTPases with EGFP-LC / A1 (Merge), and colocalization of individual Rab GTPases with EGFP-LC / A1 indicated by white dots (Colocalized). Figure 15C The Pearson colocalization coefficient (PCC) of LC / A1 with the indicated Rab3a, Rab4, Rab5, Rab7, Rab11, or Rab27a is shown. Controls used to determine colocalization with EGFP-LC / A1 (anti-GFP) and separation (DNA) are represented by dashed columns and analyzed in separate experiments (Figure 17). Data were plotted using GraphPad Prism 10.1.2. Results are the average of three replicates from three independent experiments.

[0030] Figures 16A-16B Dominant-negative Rabs involved in the SV cycle inhibit N-terminal-dependent translocation of LC / A1 to the plasma membrane. N2A cells were fixed with 4% paraformaldehyde after overnight co-transfection with pEGFP-dominant-negative Rabs and pDsRed-LC / A1 (A) or pDsRed-LC / A3 (A1-MLD) (B), and imaged for EGFP-DN Rab fluorescence (green) and DsRed-LC / A fluorescence (red). Representative images show the steady-state intracellular localization and merged maps (Merged) of individual EGFP-DN Rab (GFP-DNRab) with DsRed-LC / A1 (A) or pDsRed-LC / A3 (A1-MLD) (B). The percentage of cells containing membrane-localized DsRed-LC / A1 fluorescence alone, or DsRed-LC / A3 (A1-MLD) fluorescence alone, or in the presence of individual DN Rab, was analyzed from ten randomized fields of view across three independent experiments. Typical plasma membrane localization of DsRed-LC / A3 (A1-MLD) observed in the presence of individual DN Rab GTPase is marked with red arrows. Means and SEM were assessed using GraphPad Prism 10.1.2, and statistical analysis was performed using ordinary one-way ANOVA and Dunnett's multiple comparison test. The percentage of membrane localizations of DsRed-LC / A1 (alone) or DsRed-LC / A3V (A1 MLD) (alone) was used as a control column. ns = not significant. p<0.001.

[0031] Figures 17A-17C Colocalization and separation values ​​of Pearson colocalization coefficient (PCC) were established. (A) After overnight transfection with pEGFP-LC / A1, N2A cells were fixed with 4% paraformaldehyde, and EGFP (primary antibody: rat α-GFP IgG, followed by goat anti-rat IgG-Alexa647 (purple)) was detected, and DNA was stained (Hoechst, blue, excitation wavelength 405 nm, emission wavelength 455 nm). Representative fluorescence images are shown. (B) Representative images of EGFP-LC / A1 with α-EGFP (colocalization) or DNA (separation) were analyzed using ImageJ for colocalization, scatter plots, and Pearson colocalization coefficient (PCC) analysis, indicating separating pixels (purple / green or green / blue) and overlapping pixels (white) between the two channels, and graphs were plotted using GraphPadPrism 10.1.2, and PCC was also plotted using GraphPadPrism 10.1.2. (C) The mean PCC of at least 5 control cells was analyzed and plotted using GraphPad Prism 10.1.2.

[0032] Figure 18 A-18B. Dominant-negative Rab isolates CT / B and transferrin to distinct intracellular locations. Following overnight co-transfection with individual pEGFP-dominant-negative Rab, N2A cells were treated with (A)DsRed-cholera toxin B subunit (CT / B) or (B) transferrin at 37°C for 5 min; then fixed with 4% paraformaldehyde and imaged for EGFP fluorescence (excitation wavelength 488 nm, emission wavelength 509 nm) and CT / B (cholera toxin B subunit - Alexa-Fluor 594 nm (red)) or transferrin (transferrin - Alexa-Fluor 594 (red)). Representative images show EGFP-DN Rab GTPases (DN Rab) and CT / B-Alexa-Fluor 594 (CT / B) or transferrin - Alexa-Fluor 594 (transferrin) respectively, and their combined (merged) steady-state intracellular localization.

[0033] Figure 19A-19GThe dominant-negative Rab involved in the SV cycle inhibits N-terminal LC / A1 cleavage of SNAP-25 bound to the plasma membrane. N2A cell lysates were prepared and SDS-PAGE was performed after overnight transfection with indicated individual pEGFP-DNRab, pEGFP, and (AB) pEGFP-LC / A1 or (C) pEGFP-LC / A3V (A1-MLD). The lysates were then transferred to PVDF membranes and detected using rat α-EGFP monoclonal IgG (primary antibody: rat α-GFP) (A, C) and mouse α-SNAP-25 monoclonal IgG (primary antibody: rat α-SNAP-25) (A). Bound primary antibodies were recognized using goat anti-rat-HRP (A, C) or goat anti-mouse-HRP (A), respectively. Bound HRP-conjugated secondary antibodies were detected using SuperSignal. EGFP-LC / A1 was detected as 75-kDa protein (A1) (A,C), individual DN Rab was detected as 50-kDa protein (C), EGFP was detected as 25-kDa protein (C), or uncut (U) and cleaved (C) SNAP-25 were detected as approximately 25-kDa protein (A). (B) Data from (A) were quantified by densitometric analysis and plotted using GraphPad 10.1.2. After overnight co-transfection with plasmids encoding individual EGFPDN Rab GTPase and (D,E)EGFP-LC / A1 or (F,G)EGFP-LC / A3V (A1MLD) and the EGFP vector, N2A cells were fixed with 4% paraformaldehyde and stained with mouse anti-SNAP-25 (1-197) IgG (red, primary antibody), followed by staining with goat anti-mouse IgG (Alexa568) for cleaved SNAP-25 (residues 1-197). EGFP expression in N2A cells was measured using GFP fluorescence (10⁻¹⁰). 7 Arbitrary unit (AU), green) and cleaved SNAP-25 (cleaved SNAP-25 fluorescence (10) 7 (AU, red) Imaging. Representative N2A cells transfected with (D) EGFP-LC / A1 or (F) EGFP-LC / A3V (A1 MLD). The total fluorescence intensity of individual cells in sub-figure E is approximately 3 x 10⁻⁶. 7 AU (GFP) and 1.5X10 7AU (GFP) was used to score cleaved SNAP-25 (red) and merged SNAP-25 (EGFP (green)). The LUTs for the immunofluorescence subplots were set as follows: EGFP-A1 (D) (453 min & 3251 max), EGFP-A3V (A1MLD) (F) (299 min & 1247 max), and cleaved SNAP-25 (736 min & 1344 max) (D) (433 min & 958 max) (F). The merged channel contained LUTs based on EGFP fluorescence and cleaved SNAP-25. (E, G) Analysis of individual cells' EGFPX10 from three independent experiments. 7 AU (X-axis) and cut SNAP-25X10 7 Total fluorescence intensity (AU, Y-axis) plotted in GraphPad Prism 10.1.2. Linear regression slopes for EGFP-LC / A1 (E) or EGFP-LC / A3V (A1MLD) (G): EGFP alone (black dashed line), EGFP+EGFP-LC / A1 (purple), EGFP+EGFP-LC / A3V (A1MLD) (blue-green), and EGFP+EGFP-LC / A1+ indicating DNRab (orange). Statistical analysis was performed on the slopes of EGFP+EGFP-LC / A1 versus EGFP+EGFP-LC / A1+ indicating DNRab GTPase (E), or the slopes of EGFP+EGFP-LC / A3V (A1MLD) versus EGFP+EGFP-LC / A3V (A1MLD)+ indicating DNRab GTPase (G). ns = not significant; p<0.001 or p<0.0001.

[0034] Figures 20A-20HIntracellular localization via the N-terminus and MLD contributes to the potency and stability of LC / A, serving as a basis for duration of action. (AB) Human motor neurons were transfected overnight with mRNA encoding LC / A1, LC / A3, or LC / A3V using a two-fold serial dilution. After 48 hours, cells were washed and incubated for an indicated duration, followed by preparation of lysates, SDS-PAGE, transfer to PVDF membranes, and detection with mouse α-SNAP-25 monoclonal IgG (primary antibody: rat α-SNAP-25), an antibody that detects both LC / A-cleaved and uncleaved SNAP-25. Binding antibodies were detected using goat anti-mouse-HRP(A) and visualized with SuperSignal. Densitometry analysis of cleaved and uncleaved SNAP-25 in at least three independent experiments was plotted as a graph showing the percentage of uncleaved / cleaved SNAP-25 versus incubation time. (CE) N2A cells were transfected overnight with DNA encoding EGFP-LC / A1 (125–3.9 ng), EGFP-LC / A3 (125–3.9 ng), or pEGFP-LC / A3V (2000–62.5 ng) in two-fold serial dilutions. Cell lysates were prepared and SDS-PAGE was performed, then transferred to PVDF membranes and detected using rat α-EGFP monoclonal IgG (primary antibody: rat α-GFP) and mouse α-SNAP-25 monoclonal IgG (primary antibody: rat α-SNAP-25). The bound primary antibodies were identified using goat anti-rat-HRP (A, C) or goat anti-mouse-HRP (A), respectively, and detected using SuperSignal. Densitometry analyses of cleaved and uncleaved SNAP-25 from at least three independent experiments were normalized to total LC / A expression and plotted using GraphPad Prism 10.1.2. (Table B - Graph C) Specific activities of LC / A1, / A3, and / A3V at 30% cleavage of SNAP-25 obtained from at least three independent experiments, plotted using GraphPad Prism 10.1.2. ns = not significant. p<0.001, p < 0.001. Statistical analysis was performed using the mean from at least three independent experiments, normalized to LC / A ratio activity and protein expression per unit of transfected plasmid. (F) pEGFP-LC / A3V was engineered to generate pEGFP-LC / A3V(A1-N1), pEGFP-LC / A3V(A1-MLD), and pEGFP-LC / A3V(A1-N1, A1-MLD). In experiment (A), N2A cells were also transfected overnight with serially diluted DNA encoding pEGFP-LC / A3V(A1-N1), pEGFP-LC / A3V(A1-MLD), or pEGFP-LC / A3V(A1-N1, A1-MLD) (125–3.9 ng). Cell lysates were prepared and EGFP was detected. Cleavaged and uncleaved SNAP-25 were normalized to total LC / A expression and plotted using GraphPad Prism 10.1.2 as described in (A), with results plotted above the data in (A). In vivo duration of action of BoNT / A3 and BoNT / A3(V) was assessed. Global motor neuron deficits were monitored by (G) rotardation and local paralysis by (H) DAS test (DAS), N=5, after injection of indicated doses of BoNT / A3 or BoNT / A3(V) into the gastrocnemius muscle of mice.

[0035] Figure 21 In Neuro-2A cells, LC / A1 is transported to the plasma membrane via an intracellular pathway that cleaves SNAP-25. Following LC / A1 synthesis in the N2A cell cytoplasm, the translocation of LC / A1 to the plasma membrane and the sensitivity of SNAP-25 cleavage to DN Rab GTPases / 4, / 3a, / 27, and / 5 indicate that efficient intracellular LC / A1 transport occurs via Rab4-positive circulating endosomes to Rab3a / Rab27 synaptic vesicles, from which LC / A1 is delivered to the plasma membrane. Rab5-positive early endosomes may function by generating upstream recycle endosomes that initiate Rab4-mediated vesicle transport (solid arrows). This model allows LC / A1 to enter this pathway at various vesicles involved in the rapid SV cycle, and this pathway is sequentially linked to enable efficient movement of LC / A1 to the plasma membrane. Rab7-positive late endosomes (LE) and Rab11-positive slow REs, which are downstream of Rab-5-mediated recycling endosomes, do not participate in LC / A1 transport to the plasma membrane or SNAP-25 cleavage (dashed lines). (Image courtesy of BioRender.com) Detailed Implementation

[0036] Overview

[0037] This article discloses clostridial neurotoxin (BoNT), tetanus toxin, or SNARE cleavage homolog variants, constructs, compositions, and methods of use.

[0038] In several respects, the BoNT, tetanus toxin, or SNARE-cleaving homologs or variants thereof disclosed herein are designed to modulate the duration of action of BoNT or SNARE-cleaving homologs within target cells. Duration of action is an important characteristic of BoNT as a human therapeutic agent and a component of botulinum toxin. This document discloses BoNT variants and constructs encoding such variants, which may include amino acid substitutions, insertions, or deletions, and / or substitutions, deletions, or insertions of some or all domains to modulate the duration of action and / or potency of BoNT. For example, in some respects, as described herein, BoNT variants may include mutations and / or substitutions of some or all domains relative to the N-terminal domain and / or membrane localization domain (MLD) present in the light chain of the BoNT protein. For clarity, BoNT may be referred to individually throughout this disclosure, but it should be understood that other SNARE-cleaving homologs, such as tetanus toxin, are also included in discussions of BoNT.

[0039] In various aspects, the constructs disclosed herein provide a method for delivering BoNT light chains (LC) or variants thereof to a subject. In one or more aspects, the construct may be a synthetic mRNA construct encoding BoNT LC or variants thereof. In various aspects, BoNT LC variants may include amino acid substitutions or substitutions of some or all of the structural domains to modulate the duration of action of BoNT. Delivery of genetically engineered BoNT-LC with a specific duration of action via mRNA transfection is an alternative therapy with several advantages over currently used BoNT / A1 protein injections. For example, mRNA transfection is simpler, safer, and less expensive to produce. Furthermore, mRNA transfection for BoNT therapy carries a lower risk of inducing immune resistance compared to administration of BoNT / A1. mRNA transfection for BoNT therapy can be used in patients who have developed immunity to BoNT due to vaccination or therapeutic injection. Moreover, mRNA transfection of BoNT LC or variants thereof can be targeted through injection site, route, and engineered mRNA complexes, rather than through the binding of toxins to neuronal cell surface proteins as in current BoNT therapies. Furthermore, the mRNA constructs and methods disclosed herein allow BoNT-LC modified to cleave non-neuronal SNARE targets to target non-neuronal cells. Further, the mRNA constructs disclosed herein allow BoNT-LC to be delivered to target cells as an inhibitor of exocytosis, thereby allowing for adjustment of the duration of action based on the mRNA sequence. In all respects, any clostridium neurotoxin (or other SNARE-cleaving homolog) light chain mRNA or its truncated or extended versions may be used for therapeutic purposes and are covered herein. Additional mRNA production or delivery methods can and are intended to be used to deliver mRNA to target cells in vivo, and therapeutic use of mRNA encoding SNARE-cleaving enzymes for the treatment of diseases related to exocytosis is considered to be within the scope of this disclosure, regardless of the delivery carrier or mRNA design.

[0040] Definitions and Terms

[0041] The disclosed botulinum neurotoxin (BoNT) variants, constructs, compositions, and methods can be further described using the following definitions and terminology. The definitions and terminology used herein are for describing specific aspects only and are not intended to limit the invention.

[0042] The singular forms “a,” “an,” and “the” used in the specification and claims include the plural forms unless the context clearly specifies otherwise.

[0043] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. If certain uses of the term are not clear to those skilled in the art in the context of its use, “about” and “approximately” should be interpreted as a maximum of plus or minus 10% of that particular term, while “substantially” and “significantly” should be interpreted as a greater than or equal to 10% of that particular term.

[0044] As used herein, the terms “comprising” and “including” have the same meaning as the terms “containing” and “covering”. The terms “comprising” and “including” should be interpreted as “open-ended” transitional terms, allowing the inclusion of additional components besides those listed in the claims. The terms “comprising” and “composed of” should be understood as “closed-ended” transitional terms, disallowing the inclusion of components other than those listed in the claims. The term “substantially composed of” should be interpreted as partially closed, allowing only the inclusion of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0045] The phrase “such as / for example” should be interpreted as “for example, including”. Furthermore, the use of any and all exemplary language, including but not limited to “for example”, is intended only to better illustrate the invention and, unless otherwise stated, does not constitute a limitation on the scope of the invention.

[0046] Furthermore, when using expressions such as "at least one of A, B, and C," this structure should generally be understood in the conventional sense that can be grasped by someone skilled in the art (e.g., ， "A system having at least one of A, B, and C" includes, but is not limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together. Those skilled in the art will also understand that any separate word and / or phrase representing two or more alternative terms, whether in the specification or in the drawings, should be understood to account for the possibility of including one term, any term, or both terms. For example, the phrase "A or B" will be understood to include the possibility of including "A" or "B" or "A and B".

[0047] All expressions such as “maximum,” “at least,” “greater than,” and “less than” include the listed numbers and refer to a range that can be further subdivided into ranges and subranges. A range includes each individual member. Therefore, for example, a group with 1-3 members means a group with 1, 2, or 3 members. Similarly, a group with 6 members means a group with 1, 2, 3, 4, or 6 members, and so on.

[0048] The modal verb "may" refers to the preferential use or selection of one or more options or choices among several described embodiments or included features. When options or choices regarding a particular embodiment or a particular feature included therein are not disclosed, the modal verb "may" refers to an affirmative action regarding how to manufacture or use an aspect of the embodiment or a feature included therein, or an explicit decision regarding the use of a particular skill regarding the embodiment or a feature included therein. In the latter case, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can".

[0049] In the case of two or more nucleic acid or polypeptide sequences, the term "identical" or percentage "identity" means that two or more sequences or subsequences are identical when compared and aligned within a comparison window or specified region (measured by one of the following sequence comparison algorithms or by manual alignment and visual inspection) to obtain the maximum correspondence, or have a specified percentage of identical amino acid residues or nucleotides (i.e., having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of identity in a specified region, such as the entire nucleic acid or polypeptide sequence or its various parts or domains).

[0050] For sequence comparison, a sequence is typically used as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, the coordinates of the subsequences and the sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or other parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters.

[0051] Examples of algorithms suitable for determining sequence identity and sequence similarity percentages are the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al., respectively. Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al. J. Mol. Biol.215:403-410 (1990). Those skilled in the art will understand that software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information (NCBI). In this implementation, BLAST and BLAST 2.0, along with the parameters described herein, are used to determine the percentage of sequence identity between nucleic acids and proteins. In this implementation, the BLAST algorithm includes: firstly, identifying high-scoring sequence pairs (HSPs) by recognizing short words of length W in the query sequence (short words that match or satisfy a positive threshold score T when aligned with words of the same length in the database sequence). In this implementation, T is referred to as the adjacent word score threshold (Altschul et al., ibid.). In this implementation, these initial adjacent word hits are used as seeds to initiate a search to find longer HSPs containing them. In this implementation, the word hits extend along each sequence in both directions, as long as they can improve the cumulative alignment score. In this implementation, for nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). In this implementation, a cumulative score is calculated using a scoring matrix for each amino acid sequence. In this implementation, word matching extensions in all directions are halted when: the cumulative alignment score decreases by X from its maximum attainable value; the cumulative score becomes zero or below due to the accumulation of one or more negatively scored residue alignments; or the end of any sequence is reached. In this implementation, the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. In this implementation, the NCBIBLASTN or BLASTP program is used for sequence alignment. In this implementation, the BLASTN or BLASTP program uses the default values ​​used by NCBI. In this implementation, the default values ​​used by the BLASTN program (for nucleotide sequences) are: word length (W) of 28; expected threshold (E) of 10; maximum number of matches within the query range set to 0; match / mismatch score of 1, -2; linear gap penalty; use of a low-complexity region filter; and use only a lookup table mask. In the implementation, the default values ​​used by the BLASTP program (for amino acid sequences) are: word length (W) of 3; expected threshold (E) of 10; maximum number of matches in the query range set to 0; BLOSUM62 matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1992)); void presence penalty of 11, void extension penalty of 1; and conditional component score matrix adjustment.

[0052] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are synthetic chemical analogs of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers.

[0053] In this article, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimics are compounds whose structure differs from the general chemical structure of amino acids but whose function is similar to that of natural amino acids.

[0054] In this paper, amino acids are represented by their well-known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemistry Nomenclature Committee. Similarly, nucleotides are represented by their recognized single-letter codes.

[0055] “Conservatively modified variants” apply to both amino acid and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or substantially the same amino acid sequence, or, when the nucleic acid does not encode an amino acid sequence, substantially the same sequence. Due to the degeneracy of the genetic code, any given protein can be encoded by a large number of functionally identical nucleic acids. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where a codon designates alanine, that codon can be changed to any of the corresponding codons without changing the encoded polypeptide. This nucleic acid variation is a “silent variant,” which is a type of conservedly modified variant. Each nucleic acid sequence encoding a polypeptide described herein also describes each possible silent variant of that nucleic acid. Those skilled in the art will recognize that individual codons in a nucleic acid (except for AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, with respect to the expression product, each silent variant of the nucleic acid encoding the polypeptide is implicit in the respective described sequences, but not with respect to the actual probe sequence.

[0056] Regarding amino acid sequences, those skilled in the art will recognize that altering the sequence of a peptide, polypeptide, or protein by individual substitution of a single amino acid is a "variant of conserved modification," provided that the alteration results in the substitution of the original amino acid with an amino acid of similar chemical properties. Conservative substitutions that provide functionally similar amino acids are well known in the art. Such variants of conserved modification are complementary to, but not excluded from, polymorphic variants, interspecific homologs, and alleles.

[0057] The following eight groups each contain mutually conserved substituted amino acids: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton). Proteins (1984)).

[0058] The term "object" is used interchangeably with the terms "individual" and "patient," and includes both human and non-human objects. In some implementations, the object can be any mammal.

[0059] As used herein, the term "treatment" refers to any aspect of a patient's condition or severity that reduces, eliminates, or improves symptoms for which BoNT therapy may benefit. In various respects, this disclosure includes a method for treating a patient with symptoms for which BoNT therapy may benefit. In this context, the method may include treating the patient with an effective amount of the BoNT variant and / or mRNA construct provided herein, thereby reducing, eliminating, or alleviating symptoms.

[0060] As used herein, “therapeutic effective dose” means a dose of therapeutic agent sufficient to produce beneficial or desirable biological and / or clinical outcomes. BoNT variants and mRNA constructs may be administered at therapeutically effective doses depending on the type of treatment desired. Methods for determining appropriate doses or dose ranges for individual treatment are known to those skilled in the art. In each respect, BoNT variants and / or mRNA constructs may be administered as a single dose or, where appropriate, in multiple doses.

[0061] BoNT and other SNAREs cut homologs, variants, constructs, and combinations.

[0062] In various aspects, BoNT variants are disclosed. In some aspects, said BoNT variants can be variants of any BoNT serotype and / or subtype. As mentioned above, in some aspects, the BoNT variants disclosed herein may contain one or more amino acid substitutions, deletions, insertions, or domain substitutions that modulate the potency and / or duration of action of BoNT. In some aspects, said BoNT variants may contain one or more mutations and / or all or part of the domain substitutions relative to the N-terminus, membrane localization domain (MLD), and / or low homology domain (LHD) of the BoNT protein light chain. An example of MLD is the MLD of BoNT / A1 LC, located at amino acid residues 275-334 of the BoNT / A1 LC sequence with reference to SEQ ID NO: 3. Table 1 below lists the amino acid residues associated with these domains in various BoNTs. It should be understood that the term BoNT variant includes variants of the full-length BoNT protein and / or variants of the light chain (LC) or heavy chain (HC). In various aspects, other SNARE cleavage homologs and variants, including but not limited to tetanus toxin, are disclosed.

[0063] Table 1: N-terminal domains, MLD, and LHD of various BoNTs

[0064] As described herein, in various respects, BoNT variants and BoNT LC variants may include one or more substituted domains. A non-limiting list of potential substituted domains in BoNT includes the N-terminus, MLD, and LHD. As used herein, a substituted domain refers to the removal of a wild-type domain (e.g., the MLD domain) of a reference BoNT and its replacement with an MLD from another BoNT. As an example using Table 1 above, amino acid residues 275-334 (MLD) of BoNT / A1 may be removed and replaced with amino acid residues 277-334 (MLD) of BoNT / A3, thereby providing a BoNT / A1 variant comprising a substituted MLD from BoNT / A3.

[0065] The duration of BoNT action can be determined in various ways, for example, in vivo using mouse models and / or in vitro using cultured cells. Both methods are described with reference to Examples 2 and 3 below, and results show… Figure 9 , 10In 11, for example, in an in vivo mouse model, the duration of BoNT action can be assessed using the robin test and / or toe abduction scoring test. In vitro determination of the duration of BoNT activity can be assessed by measuring uncut and / or cleaved SNARE proteins. In each aspect, cleaved or uncut SNARE proteins, such as VAMP, SNAP-25, and syntaxin, can be measured. In one aspect, the in vitro determination of the duration of BoNT activity can be assessed by measuring uncut and / or cleaved SNAP-25 in ruptured neurons.

[0066] In one exemplary aspect, the BoNT / A3 protein may include an N-terminus, MLD, and / or LHD derived from the BoNT / A1 protein, which may shorten the duration of BoNT action. In some aspects, the BoNT / A3 variant may comprise or consist of a heavy chain, and further include a light chain having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In various aspects, the BoNT / A3 variant may comprise or consist of a light chain, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 11.

[0067] In various aspects, the BoNT / A1 protein (or tetanus toxin (TT) protein, or a homolog of BoNT or TT or other SNARE-cleaved homologs) may include the N-terminus, MLD, and / or LHD of the BoNT / A3 protein, which may prolong the duration of BoNT action. In some aspects, the BoNT / A1 variant may comprise or consist of a heavy chain and also include a light chain having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 9. In various aspects, the BoNT / A1 variant may comprise or consist of a light chain having an amino acid sequence that is the amino acid sequence of SEQ ID NO: 9.

[0068] In various aspects, any BoNT protein can be modified to include the N-terminus, MLD, and / or LHD from BoNT / A1, which may help prolong the duration of BoNT action. For example, in one aspect, the N-terminal domain, MLD, and / or LHD of the BoNT / E protein can be replaced with the N-terminus, MLD, and / or LHD from BoNT / A1, which allows for a prolonged duration of BoNT action. In some aspects, the N-terminus, MLD, and / or LHD of the BoNT / F protein can be replaced with the N-terminal domain, MLD, and / or LHD from BoNT / A1, which allows for a prolonged duration of BoNT action. Wild-type BoNT / E and BoNT / F proteins enter cells faster than BoNT / A1, but wild-type BoNT / E and BoNT / F exhibit a shorter duration of BoNT action. Therefore, by modifying BoNT / E and / or BoNT / F proteins with the N-terminus, MLD, and / or LHD of BoNT / A1, BoNT variants with rapid cell entry and prolonged duration of action can be generated. It should be understood that other BoNT serotypes can also be modified to include the N-terminus, MLD, and / or LHD of the BoNT / A1 protein, thereby modulating and / or increasing the duration of BoNT action, such as BoNT / A2, A4, A5, A6 proteins, or serotypes B, C, D, and G. Alternatively, the N-terminus, MLD, and / or LHD of any BoNT protein may contain any amino acid substitutions, deletions, or insertions to modulate the duration of BoNT action.

[0069] In each respect, the BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F and / or BoNT / G proteins or whole toxins may include replacing their N-terminus, MLD and / or LHD with the corresponding N-terminus, MLD and / or LHD from BoNT / A1 or BoNT / A3 to modulate potency and / or duration of action.

[0070] In various aspects, BoNT / B, BoNT / D, BoNT / F, and / or BoNT / G proteins or holotoxins may include replacing their N-terminus, MLD, and / or LHD with the corresponding N-terminus, MLD, and / or LHD of one or more other BoNT / B, BoNT / D, BoNT / F, and / or BoNT / G proteins or holotoxins. For example, BoNT / B holotoxin may include replacing its N-terminus, MLD, and / or LHD domains with the N-terminus, MLD, and / or LHD from BoNT / D, BoNT / F, or BoNT / G.

[0071] In various respects, as described above, this document considers a variety of BoNT light chain (LC) variants. For example, in some respects, this document also considers BoNT / A3 light chain (LC) variants that do not contain a heavy chain, which may include an N-terminus, MLD, and / or LHD from BoNT / A1. In some respects, BoNT / A3 LC variants may contain or consist of a heavy chain, and further include a light chain having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In various respects, BoNT / A3 LC variants may contain or consist of a light chain whose amino acid sequence is the amino acid sequence of SEQ ID NO: 11.

[0072] In some respects, BoNT / B light chain (LC), BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC and / or BoNT / G LC may contain the N-terminus, MLD and / or LHD replaced by the corresponding N-terminus, MLD and / or LHD of BoNT / A1 or BoNT / A3.

[0073] In several aspects, the BoNT / B LC, BoNT / D LC, BoNT / F LC, and / or BoNT / G LC may include replacing its N-terminus, MLD, and / or LHD with the corresponding N-terminus, MLD, and / or LHD of one or more of the remaining BoNT / B, BoNT / D, BoNT / F, and / or BoNT / G. For example, the BoNT / B LC may include replacing the N-terminus, MLD, and / or LHD domains with the N-terminus, MLD, and / or LHD from BoNT / D, BoNT / F, or BoNT / G.

[0074] In each respect, the BoNT or SNARE cleaving homologous light chain or its variants may be the light chain of BoNT / E or its variants, or the light chain of BoNT / F or its variants, or the light chain of BoNT / B or its variants, or the light chain of BoNT / C or its variants, or the light chain of BoNT / D or its variants, or the light chain of BoNT / G or its variants, or the light chain of BoNT / Wo or its variants, or the light chain of BoNT / Ef or its variants, or the light chain of BoNT / X or its variants, or the light chain of TeNT or its variants, or the light chain of any other member of the SNARE cleaving toxin family.

[0075] In one or more aspects, BoNT / A3 LC variants are disclosed, which include amino acid substitutions in the N-terminal region (e.g., Q7P and V14G–SEQ ID NO: 7). It should be understood that the N-terminal mutations of the BoNT / A3 LC mentioned herein can also be present in the full-length BoNT / A3.

[0076] In various aspects, heavy-chain-free BoNT / A1 light chain (LC) variants are also considered, which may comprise MLD and / or LHD from BoNT / A3. In some aspects, the BoNT / A1 LC variant may comprise or consist of a heavy chain, and further comprise a light chain having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 9. In various aspects, the BoNT / A1 LC variant may comprise or consist of a light chain, the amino acid sequence of which is the amino acid sequence of SEQ ID NO: 9.

[0077] The BoNT variants, including the BoNT LC variant, can be produced in various ways. For example, the BoNT variants and / or BoNT LC variants can be produced recombinantly in Escherichia coli, yeast, or other suitable expression systems.

[0078] As described above, synthetic mRNA constructs are disclosed in various aspects. In various aspects, the synthetic mRNA constructs are used to generate LCs of clostridial neurotoxins (e.g., BoNT, TT, or homologs thereof). In various aspects, the synthetic mRNA constructs are used to generate BoNT LCs or LC variants thereof. In some aspects, the mRNA constructs may encode any BoNT LC or LC variant disclosed herein. In various aspects, the mRNA constructs may encode any BoNT LC, TT, or TT LC, or homologs of BoNT or TT.

[0079] In all respects, the mRNA construct may encode a BoNT LC or LC variant having an amino acid sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 99%, or 100% identical to one or more of SEQ ID NO: 3, 5, 7, 9, 11, 31, 16, 19, 22, 13, 25, or 28. It should be understood that the mRNA construct may encode any BoNT LC variant, or TT variant, or homolog of TT or BoNT, for example, an LC variant having one or more amino acid substitutions, insertions, or deletions, and / or substitutions, deletions, or insertions of partial or complete domains, to modulate the duration and / or potency of BoNT or TT action.

[0080] In various aspects, the mRNA construct may include one or more features to customize transfection efficiency. For example, in one aspect, the mRNA construct may include a 150-nucleotide poly(A) tail and / or a Cap 1 upgrade. In the same or alternative aspects, the mRNA construct may have reduced dsRNA content. In some aspects, the mRNA construct may be customized to target neuronal cells. In alternative aspects, the mRNA construct may be customized to target other cell types, including but not limited to cancer cells and / or cells associated with inflammatory processes.

[0081] In each aspect, the mRNA construct can be formulated and / or present in lipid nanoparticles. In each aspect, the lipid nanoparticles can be any lipid nanoparticle suitable for the mRNA constructs and methods disclosed herein. In one aspect, the lipid nanoparticles can be negatively charged, positively charged, or both.

[0082] In various aspects, compositions are disclosed herein. For example, in some aspects, the compositions may include one or more mRNA constructs disclosed herein. In some aspects, the compositions may include one or more mRNA constructs formulated and / or present in lipid nanoparticles. In alternative aspects, the compositions may include BoNT variants and / or BoNT LC variants disclosed herein. In one or more aspects, the compositions may also include one or more suitable pharmaceutically acceptable carriers. In some aspects, the suitable pharmaceutically acceptable carriers may include RNase-free water, phosphate-buffered saline, etc.

[0083] This disclosure also provides a kit. In some aspects, the kit may include a formulation of the BoNT variant, BoNT LC variant, mRNA construct, or composition disclosed herein, along with instructions for dosing. In one or more aspects, the kit may be used in the methods disclosed herein.

[0084] method

[0085] In various aspects, the methods disclosed herein may include methods of administering a BoNT variant or a BoNTLC variant to a subject in need. In some aspects, the subject in need may be a subject with symptoms requiring BoNT treatment. In various aspects, the methods disclosed herein may include treating a patient and / or subject with a therapeutically effective amount of a BoNT variant or a BoNTLC variant. In such aspects, treatment may include administering a therapeutically effective amount of a BoNT variant or a BoNTLC variant to a patient and / or subject. In another aspect, the treatment is a cosmetic treatment. It should be understood that the treatments contemplated in this disclosure include not only treatments that produce a therapeutic effect in neuronal cells, but also treatments performed in any type of cell where inhibition of exocytosis may be desirable, including but not limited to inflammatory states, skin diseases (such as acne), allergies, certain metabolic diseases, hormonal disorders, and cancer. In one aspect, a BoNT variant or a BoNTLC variant may be administered for targeted therapy in cancer cells and / or cells associated with inflammatory processes. It should be understood that administering a BoNTLC variant may include administering an mRNA construct for generating the BoNTLC variant.

[0086] In some respects, the BoNT variants and / or the BoNT LC variants can be administered in any suitable manner. In one or more respects, the BoNT variants and / or the BoNT LC variants can be administered topically or by injection, such as intramuscular or subcutaneous injection.

[0087] In all respects, the BoNT variants and BoNT LC variants may include any or all of the features discussed above regarding the BoNT variants and BoNT LC variants.

[0088] In some aspects, one or more mRNA constructs, formulations, and / or compositions may be administered to a patient and / or subject to perform one or more of the treatments described above. In one or more aspects, the mRNA construct may encode one or more BoNT variants and / or BoNT LC variants disclosed herein. In some aspects, BoNT variants and / or BoNT LC variants may be administered as one or more mRNAs encoding one or more BoNT variants and / or BoNT LC variants.

[0089] There are no particular restrictions on the timing and dosage of administration, and these can vary depending on the severity of symptoms, age, sex, weight, site of administration, and route of administration.

[0090] Example

[0091] The following embodiments are illustrative and are not intended to limit the scope of the claimed subject matter.

[0092] Example 1 - Botulinum neurotoxin A1 light chain migrates to the Neuro-2A cell membrane via the synaptic vesicle circulation pathway. SNAP-25 on

[0093] Brief

[0094] Botulinum neurotoxin type A (BoNT / A) is a potent neurotoxin and a treatment for neurological disorders in humans. The 150 kDa BoNT / A contains a catalytic light chain (LC / A) linked to the heavy chain via disulfide bonds, enabling it to enter host cells. The molecular mechanisms involved in BoNT / A migration to the plasma membrane to target SNAP-25 are relatively poorly understood aspects of BoNT poisoning. BoNT / A comprises eight subtypes (1–8), and previous data have shown that LC / A1 and LC / A3 have different migration pathways and intracellular localizations. This study further investigated intracellular LC / A migration in neurons, leveraging our understanding of the role of Rab proteins in synaptic vesicle recycling. Imaging studies revealed co-localization of LC / A1 with endogenous Rab GTPases and identified dominant-negative Rab GTPases that block LC / A1 plasma membrane localization, suggesting that LC / A1 migrates to the plasma membrane via the synaptic vesicle recycling pathway. Mass spectrometry analysis confirmed the co-localization of Rab GTPases with intracellular LC / A3. The discovery of this synaptic vesicle circulation pathway targeting LC / A1 to the plasma membrane identifies a new host protein target for BoNT / A therapy to alleviate the intracellular effects of BoNT.

[0095] introduction

[0096] Clostridium botulinum is a Gram-positive anaerobic bacterium that produces botulinum neurotoxins (BoNTs), which are the most toxic proteins to humans. BoNTs are 150 kDa zinc-dependent metalloproteinases[1] with a 50-kDa N-terminal catalytic light chain (LC) and a 100 kDa C-terminal heavy chain (HC). The HC contains an LC translocation domain (HCN) and a receptor-binding domain (HCC), which enable neuronal cell binding and subsequent endocytosis and translocation of the LC to the cytoplasm[2-4]. BoNTs include seven pathogenic serotypes (AG); BoNT / A includes eight subtypes (BoNT / A(1-8))[5, 8], which specifically cleave the 25 kDa synaptosome-associated protein (SNAP-25) on the cytoplasmic side of the neuronal plasma membrane[5].

[0097] The process by which BoNT / A enters the neuronal cytoplasm from the cell surface has been well studied. BoNT / A binds to gangliosides on the surface of resting neurons, followed by the receptor protein synaptic vesicle protein-2 (SV2) exposed on the cell surface during synaptic vesicle (SV) exocytosis [6]. The BoNT / A-receptor complex is endocytosed during the reabsorption of SV from the plasma membrane. After SV matures, it either fuses with acidic endosomes or undergoes direct luminal acidification and neurotransmitter reloading. In response to acidification, the HC of BoNT / A N Insertion into the synaptic vesicle membrane and facilitating the light chain's crossing of the SV membrane and translocation into the neuronal cytoplasm [7]. Studies have shown that in the cytoplasm, the disulfide bond between LC and HC is reduced by the thioredoxin reductase system with the assistance of the heat shock protein HSP90, thereby releasing the light chain into the cytoplasm. However, the molecular process that guides intracellular LC / A1 to its enzymatic target (i.e., SNAP-25 bound to the plasma membrane) is a less studied but crucial aspect of BoNT poisoning.

[0098] Recent studies have shown that the light chains of BoNT / A1 and BoNT / A3 are located in different intracellular regions, with LC / A1 located in the host cell membrane and LC / A3 located in intracellular vesicles. BoNT / A1 has higher potency than BoNT / A3, suggesting that membrane binding may be related to potency [8]. Through studies of LC / A1, LC / A3, and the N-terminal variant of LC / A3 known as LC / A3V, two regions within LC / A1 have been shown to be responsible for the intracellular migration of LC / A1 to the plasma membrane: one is the N-terminus that binds to intracellular vesicles (called A1N, residues 1-17), and the other is the internal region of LC / A1 that directs cytoplasmic target proteins to the plasma membrane (called the membrane localization domain (MLD), residues 275-334) [8-11]. This study utilized a set of Rab proteins for co-localization studies to further characterize the intracellular migration pathways of LC / A1, LC / A3, and LC / A3V in Neuro-2A (N2A) cells. Data indicate that LC / A1 utilizes the synaptic vesicle circulation pathway to target SNAP-25 on the plasma membrane.

[0099] result

[0100] Microtubules are involved in the binding of BoNT LC to intracellular vesicles. To investigate the role of cytoskeleton structures in the intracellular migration of LC / A1 cells, the effects of two cytoskeleton inhibitors on the intracellular localization of LC / A1 and LC / A3 cells in Neuro-2a cells were tested. Neuro-2a cells were first transfected with GFP (cytoplasmic localization), GFP-LC / A1 (plasma membrane localization), GFP-LC / A3 (vesicle localization), or GFP-LC / A3V (cytoplasmic localization with nuclear exclusion features), and fusion proteins were expressed before treatment with cytoskeleton inhibitors. Cytochalasin D (CD) binds to the polymeric ends of actin filaments, thereby disrupting elongation, inhibiting growth, and causing actin filament aggregation [12-17]. Phalloidin staining of CD-treated N2A cells showed actin disruption, while β-tubulin staining showed microtubule-stained neurite extension ( Figure 1 A), but this did not disrupt the localization of exogenously expressed EGFP-LC / A1 on the plasma membrane or the localization of LC / A3 on synaptic vesicles (SVs). Real-time fluorescence imaging showed that in CD-treated N2A cells, EGFP-LC / A1 was localized on the membrane and moved along neurites (A). Figure 1 B). Therefore, EGFP-LC / A1 localizes to the plasma membrane and migrates along neurites independently of actin.

[0101] Nocodazole binds to β-tubulin, preventing its binding to α-tubulin, thereby causing microtubule depolymerization. This can be used to investigate the role of microtubules in LC / A1 migration [18,19]. In nocodazole-treated N2A cells, exogenously expressed EGFP-LC / A1 remained in a membrane-localized state, while LC / A3 changed from a vesicle-binding protein to a cytoplasmic protein (…). Figure 1 A) indicates that nocodazole disrupts microtubules and disperses LC / A3 into the cytoplasm. These studies suggest that microtubules play a role in the intracellular migration of LC / A cells.

[0102] Colocalization of intracellular LC / A1 with Rab GTPases in N2A cells. Intracellular transport utilizes Rab GTPases, which are distributed in different compartments to facilitate the phased transport of intracellular vesicles

[20] . Therefore, the colocalization of Rab GTPases with LC was characterized to determine the vesicle properties associated with intracellular LC / A1. Preliminary experiments used Pearson correlation coefficient (PCC) to detect the colocalization of Rab:LC / A1 in Neuro-2a cells

[21] . The maximum colocalization value was set by measuring the colocalization of EGFP-LC / A1 with EGFP-bound EGFP antibody (0.8 PCC), while the minimum colocalization value was set by measuring the colocalization of EGFP-LC / A1 with Hoechst-stained DNA (-0.1 PCC). Figure 5 Exogenously expressed EGFP-LC / A1 under steady-state conditions showed the highest colocalization with Rab4 and Rab27a, lower colocalization with Rab5, Rab7, Rab11, and Rab4, and limited overall colocalization with Rab3a (Fig. 2). Examination of overlapping image pixels revealed that EGFP-LC / A1 colocalizes with Rab3a and Rab27a along the plasma membrane, with Rab4, Rab5, and Rab11 intracellularly, and with Rab7 in the perinuclear region (Fig. 2). Overall, the steady-state colocalization data suggest that LC / A1 migration is associated with Rab GTPases involved in multiple intracellular functions.

[0103] Dominant-negative (DN) Rab GTPases block the plasma membrane localization of LC / A1. To identify the Rab GTPases that regulate the intracellular migration of LC / A1 to the plasma membrane, DNA encoding individual EGFP-DN Rab GTPases was co-transfected with pDsRed-LC / A1, and the intracellular distribution of DsRed-LC / A1 was measured (Fig. 3). DN Rab GTPases preferentially bind GDP, thereby blocking Rho GEF and stopping Rab-directed vesicle movement. Therefore, DN Rab GTPases can be used to identify Rab GTPases that play a role in the migration of LC / A1 to the plasma membrane [22-24]. Co-transfection of EGFP-LC / A1 and DsRed-LC / A1 showed co-localization of the two fluorophores. DsRed-LC / A1 was co-transfected with DNA encoding DN Rab GTPases known to play a role in anterograde and antidrograde migration to and from the plasma membrane [25-27], including DN Rab3a, DN Rab4, DN Rab5, and DN Rab27b. DN Rab 3a, 4, 5, and 27b were localized in the cytoplasm and isolated LC / A1 from the plasma membrane (Fig. 3). The intracellular localization of LC / A1 varied among the positively correlated DN Rabs. When co-expressed with DN Rab3a or Rab5, LC / A1 was located close to the plasma membrane; while when co-expressed with DN Rab4 or Rab27b, which are located upstream of Rab3

[26] , LC / A1 was located close to the perinuclear region (Fig. 3). Rab27a and Rab27b are isoforms

[28] . The difference lies in that co-expression of DN Rab7 or Rab11 did not alter the plasma membrane localization of LC / A1, indicating that neither the Rab7 nor Rab11 pathways were used for LC / A1 migration to the plasma membrane. Overall, the DN Rab GTPase that inhibits LC / A1 binding to the plasma membrane plays a role in the synaptic vesicle (SV) reabsorption pathway, suggesting for the first time that LC / A1 utilizes the synaptic vesicle reabsorption pathway for anterograde migration to the plasma membrane.

[0104] A control experiment was conducted to test the effects of DN Rab GTPase on the endocytosis of cholera toxin B subunit (CT / B) and transferrin, as previously described [29,30]. Although Rab4

[31] and Rab11

[32] are involved in transferrin migration, the expression of DN Rab4 or DN Rab11 did not inhibit transferrin and had limited effect on intracellular uptake and regeneration of transferrin in N2A cells, consistent with previous findings [33,34].

[0105] Intracellular LC / A3 is associated with Rab GTPases involved in multiple vesicle transport pathways. To detect the interaction between intracellular LC / A and Rab GTPases, the association of Rab GTPases with exogenously expressed EGFP, EGFP-LC / A3, or EGFP-LC / A1 was measured by mass spectrometry (MS). N2A cells were lysed and postnuclear supernatant (PNS) containing soluble proteins was prepared by centrifugation. MS analysis identified 29 Rab GTPases in the PNS of control (untransfected) N2A cells (Supplementary Tables 2-3). Rab proteins associated with LC / A were identified by immunoprecipitation from PNS and LC-MS / MS. MS assessment showed that the enrichment of Rab GTPases in LC / A3-IP was higher than that in LC / A1-IP (Table 2), which is consistent with the homeostatic localization of LC / A3 bound to intracellular vesicles and LC / A1 mainly associated with plasma membrane[8]. Pretreatment of PNS with nocodazole before IP increased the recovery of LC / A3 and LC / A1, consistent with their intracellular microtubule binding. Rab1a, Rab2 (a / b), Rab3 (a / c), Rab5b, Rab6a, Rab7a, Rab10, Rab11b, Rab14, and Rab21 were enriched more than 2-fold in EGFP-LC / A3 immunoprecipitation (Table 2). Although DN Rab4 and DN Rab27a inhibited LC / A1 migration to the plasma membrane, Rab4 and Rab27a were not found in EGFP-LC / A3 or LC / A1 immunoprecipitation (Figure 3). Since the N2A proteome confirmed the presence of Rab4 and Rab27a in the postnuclear supernatant, we conclude that only a small fraction of total Rab4 and Rab27a interact with LC / A3 or LC / A1 (Table 3). Overall, the Rab GTPases co-precipitated with LC / A3 suggest that LC / A interacts with synaptic vesicle recycling pathways, late endosomes, and endoplasmic reticulum (ER) / Golgi apparatus transport.

[0106] Table 2. Rab GTPases in control or nocodazole-treated N2A cells, immunoprecipitated with EGFP, EGFP-LC / A1, or EGFP-LC / A3LM.

[0107]

[0108] Table 3: Neuro-2A Rab proteome

[0109] discuss

[0110] This study developed a transfection-based model to investigate the intracellular migration of exogenously expressed LC / A1 to the plasma membrane [8, 11]. Preliminary experiments suggested a role for microtubules in the migration of LC / A1 along intracellular vesicles. Next, steady-state imaging revealed that LC / A1 co-localizes with multiple Rab GTPases promoting various cellular functions, including migration to the plasma membrane and protein degradation. These data were further confirmed by co-expression of LC / A1 with a group of DNA Rab GTPases, suggesting that the SV cycle pathway is involved in LC / A1 migration to the plasma membrane. Finally, intracellular LC / A1 and LC / A3 were co-precipitated. Combined with mass spectrometric evaluation of the co-precipitated proteins, a comprehensive assessment of the Rab GTPase-LC / A1 interaction was provided, revealing its association with Rab GTPases involved in both the SV cycle pathway and intracellular degradation pathways. This is the first time that LC / A has been shown to migrate via an intracellular vesicle pathway, thereby achieving stable co-localization with SNAP-25 on the plasma membrane, indicating that LC / A1 migrates via both anterograde and retrograde pathways, leading to SNAP-25 targeting and light chain degradation

[45] . This method can be used to measure the migration of the catalytic domains of other proteotoxic agents to their intracellular substrates, which is a less studied aspect of proteotoxic agent action.

[0111] In the presence of nocodazole and cytochalasin D, LC / A1 maintains membrane localization, while in nocodazole-treated N2A cells, LC / A3 diffuses into the cytoplasm. Figure 1A). Nocodazole affects LC / A3 but not LC / A1, possibly because LC / A1 is stably bound to the intracellular plasma membrane and SNAP-25 at homeostasis, while LC / A3 is mainly located in vesicles and cytoplasm, thus its migration to the plasma membrane can still be blocked. Therefore, LC / A1, and LC / A in the common sense, may also utilize microtubules for intracellular migration to the plasma membrane. Microtubules play a crucial role in many cellular activities, including cell division, transport, and movement. For this reason, microtubules are frequently utilized by bacterial toxins. Our study examined the homeostatic level of intracellularly expressed LC / A, and the pathway of natural BoNT toxicity in neurons involves a multi-step entry process. Prior to endocytosis, BoNT / A binds to gangliosides and SV proteins on the neuronal surface [35, 36]. BoNTs mainly enter cells via a clathrin-dependent pathway in an intravesical form [37-39]. Studies on BoNT-HC entry into N2A cells have shown that nocodazole treatment reduces HC binding, while genistein (an inhibitor of pit-mediated endocytosis and actin network) does not reduce HC binding [40, 41]. Combined with our data, these studies suggest that BoNT / A utilizes microtubules for entry and intracellular migration. In N2A cells treated with cytochalasin D (leading to actin filament aggregation), real-time imaging showed LC / A1 moving along microtubules in neurite buds (…). Figure 1 B).

[0112] Although previous experiments have characterized the contribution of Rab GTPases in BoNT poisoning [42-44], this study measured the intracellular migration of exogenously expressed EGFP-LC / A1 colocalized with endogenous Rab proteins, as well as in the presence of DN Rab proteins, and finally examined the direct interaction of endogenous Rab GTPases by co-immunoprecipitation assays of LC / A1 and / A3, suggesting that LC / A1 migrates to the plasma membrane via the synaptic vesicle circulation pathway (Figure 3).

[0113] Several Rab GTPases colocalize with LC / A1, and several DN Rab GTPases inhibit the migration of LC / A1 to the plasma membrane. DN Rab5 inhibits the migration of LC / A1 to the plasma membrane, holding LC / A1 on intracellular vesicles (Fig. 3). Rab5(ac)

[45] mediates clathrin-dependent endocytosis, the main pathway for the recovery of synaptic vesicles from the plasma membrane [27,46,47]. In Drosophila, DNRab5 mediates isomorphic fusion of synaptic vesicles, increases synaptic vesicle size, and inhibits neurotransmitter release

[48] . In addition, DN Rab5 inhibits early endosome fusion

[49] . As observed in ExoS

[50] , DN Rab5 does not colocalize with LC / A1, while Rab5 shows moderate colocalization, suggesting that DN Rab5 blocks the formation of sorting endosomes either by holding bound LC / A1 upstream of sorting endosomes or by preventing migration to the recovery endosomes due to the lack of sorting endosomes. LC / A1 showed greater colocalization with Rab4 than with Rab11, and DN Rab4 (but not DN Rab11) inhibited LC / A1 migration to the plasma membrane (Fig. 3). Rab4 and Rab11 promote endosome resorption prior to SV formation

[51] . Kinesin-2 mediated endosome resorption is divided into two groups: Rab4-mediated rapid resorption and Rab4-to-Rab11-mediated slow resorption [51-53]. Therefore, LC / A1 may utilize the Rab4-mediated rapid resorption route to reach the plasma membrane. In addition, LC / A1 was observed to have a high degree of colocalization with Rab27b, but little colocalization with Rab3a (Fig. 2). However, expression of either DN Rab3a or DN Rab27b inhibited LC / A1 migration to the plasma membrane (Fig. 3). In neurons, Rab3a and Rab27b control docking, fusion, and exocytosis of synaptic vesicles

[51] . Rab3(ad)

[54] is crucial for kinin-1-mediated rapid anterograde migration and vesicle assembly on microtubules

[55] . Although Rab3(bd) has functional redundancy, knockout models show that removal of Rab3a leads to postnatal death

[56] . Rab3 and Rab27(ab)

[28] are structurally related, partially co-localized, and share effector proteins such as Rabphilin-3A, which binds to both Rab3 and Rab27 and regulates synaptic vesicle cycling before migration to the plasma membrane and localization to SNAP-25[57,58].[59-61]. Although present in overlapping pools, Rab3 and Rab27 transition to different synaptic vesicles over time

[57] , which could explain why expression of either DN Rab3a or DN Rab27b inhibits LC / A1 migration to the plasma membrane, but with a greater degree of co-localization with Rab27b. DN Rab7 does not interfere with LC / A1 migration to the plasma membrane (Fig. 3).The association of LC / A1 with Rab7 and other Rab GTPases involved in reverse protein transport may reflect a common interaction between LC / A1 and intracellular vesicles that are involved in the reverse and anterosynaptic vesicle reuptake pathways that transport LC / A1 to the plasma membrane (Table 2).

[0114] Using data from this study, research on Rab GTPase activity [26,27], and previous data on BoNT migration and LC / A localization [8,40,62], we propose a BoNT / A intracellular migration model ( Figure 4 Initially, BoNT / A1 binds gangliosides and SV-2 exposed on the surface in an activity-dependent manner. The bound BoNT / A1 is internalized into the early endosome (Rab5) and migrates retrogradely along microtubules to the perinuclear region, where synaptic vesicles recycle the endosome for maturation, leading to luminal acidification and light chain translocation across the vesicle membrane. In the cytoplasm, LC / A1 is released via disulfide bond reduction, refolds, and binds to Rab4-positive synaptic vesicles via its N-terminus (N), thereby migrating anterogradely along microtubules and converting to Rab3a-Rab27b vesicles. Upon adhering to the plasma membrane, LC / A1-MLDs stably bind to SNAP-25 for cleavage.

[0115] While a single DN Rab GTPase typically suggests the use of a particular signaling pathway

[63] , Rab GTPases can have specialized functions within neurons

[27] . This study used three approaches to define and validate multiple Rab GTPases that regulate known synaptic vesicle transport pathways, covering processes from early LC / A1 endosome interactions to stable binding to SNAP-25 on the plasma membrane, suggesting the dynamic nature of LC / A intracellular migration and eventual degradation. Understanding the kinetics of BoNTLC intracellular migration may facilitate interventions targeting host proteins to mitigate BoNT toxicity and contribute to the development of novel drugs.

[0116] Materials and methods

[0117] Unless otherwise stated, all reagents were purchased from Life Technologies (Grand Island, NY, USA).

[0118] EGFP-LC / A construct and Rab dominant-negative variants were engineered. As previously described [8], DNA encoding A1 (1-4450), A3V (1-446), and A3LM (1-446) from plasmid pEGFP-C3 was engineered. Enhanced green fluorescent protein (EGFP) was subcloned into the SacI-BamHI restriction site of pEGFP-C3 as a fusion of LC / A1, LC / A3V, LC / A3LM, and Rab GTPases 3a, 4, 5, 6, 11, and 27a. Primers were designed and obtained from New England Biolabs® NEBaseChanger® (Ipswich, Massachusetts, USA) to engineer the dominant-negative variants of the Rab protein: Rab3a(T 36 N), Rab4 (S) 22 N), Rab5 (S) 34 N), Rab7 (T) 22 N), Rab11 (S) 25 N) and Rab27b (T 23 N).

[0119] Cell culture. Neuro-2A (N2A) cells were obtained from ATCC (Manassas, Virginia, USA) CCL-131. N2A cells were cultured in a complete essential medium supplemented with 10% fetal bovine serum, 1x penicillin-streptomycin, 0.1% sodium bicarbonate, 1 mM sodium pyruvate, and 1% non-essential amino acids at 37°C in a humidified 5% CO2 (v / v) environment.

[0120] N2A cell transfection and cytoskeleton inhibitor treatment. N2A cells were transfected as previously described

[64] . N2A cells were seeded at a density of 50,000 cells / well in 24-well plates on acid-etched glass coverslips coated with poly-D-lysine (1:500). The following day, N2A cells were transfected with 500 ng of the specified plasmid (Lipofectamine LTX; Invitrogen) as previously described. ™ (Waltham, Massachusetts, USA)[8]. After overnight transfection, cells were treated for 60 minutes with 4 µM cytochalasin D (C8273, Sigma-Aldrich, St. Louis, USA) or 9 µM nocodazole (M1404, Sigma-Aldrich, St. Louis, USA).

[0121] Immunofluorescence imaging (IF). After transfection, N2A cells were fixed with 4% (w / v) paraformaldehyde for 15 minutes at room temperature. N2A cells were then washed twice with room temperature PBS and plated in DBPS (14040182, Gibco) containing 10% FBS (v / v), 2.5% cold-water fish skin gelatin (w / v), 0.1% Triton-X (v / v), and 0.05% Tween-20 (v / v). ™ (In Illinois, USA) closed for 60 minutes. N2A cells were incubated with primary antibodies: 1:5000 rabbit α-β-3 tubulin (ab52623, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab3a (ab3335, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab4 (ab109009, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab5 (ab218624, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab7 (ab137029, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab11 (ab180504, Abcam, Cambridge, UK) or 1:1000 rabbit α-Rab27a (ab223044, Abcam, Cambridge, UK), in a solution containing 5% FBS (v / v), 1% cold-water fish skin gelatin (w / v), and 0.1%... Incubate overnight at 4°C with shaking in DBPS (incubation solution) containing Triton-X (v / v) and 0.05% Tween-20 (v / v). The next day, N2A cells were washed three times (5 min each) with DPBS containing 0.005% Tween-20 (v / v) and incubated for 60 min at room temperature with complementary Alexa-Fluor-conjugated secondary antibody or phalloidin-Alexa-fluor 568 nm (1:250) (A12380, LifeTechnologies, Wisconsin, USA). Cells were then washed twice with 0.005% Tween-20 (v / v) DPBS and incubated with Hoechst (1:10,000 in DPBS, H21492, Invitrogen). ™ Stain for 5 minutes with a microscope slide (Invitrogen, Massachusetts, USA). Flip the coverslip onto the microscope slide and cure with 8 µL Prolong™ Gold anti-fluorescence quenching mounting medium (Invitrogen, Massachusetts, USA).

[0122] In addition, N2A cells were washed twice with DPBS at 4°C and stained with wheat germ lectin (WGA) (1:1000, W32466, Invitrogen, Massachusetts, USA) as a membrane dye at 4°C for 30 minutes. After WGA incubation, N2A cells were washed twice with DPBS, then fixed with 4% (w / v) paraformaldehyde at room temperature for 15 minutes, washed twice with DPBS, and stained with Hoechst (1:10,000 in DPBS, H21492, Invitrogen). ™ The sample (from Waltham, Massachusetts, USA) was treated as a nuclear marker at room temperature for 5 minutes. The coverslip was then flipped onto a microscope slide and cured with 8 µL Prolong™ Gold anti-fluorescence quenching mounting medium (Invitrogen, Waltham, Massachusetts, USA).

[0123] Live-cell imaging. N2A cells were cultured and analyzed as previously described [8, 64]. As described above, N2A cells were treated with 4 µM cytochalasin D and incubated for 60 min four hours after transfection with LC / A1. Live-cell imaging of N2A cells was performed using a 60x oil immersion objective (1.4NA objective) on a Nikon Eclipse Ti inverted microscope with the stage heated to 37°C (Frank E. Fryer A-50), and data analysis was performed using Eclipse software. Images were acquired every 15 seconds for 15 minutes. Videos were compiled using Nikon Elements AR 1.60.00 64-bit software (Melville, NY, USA) and Image J [65, 66].

[0124] EGFP-LC / A immunoprecipitation. N2A cells were seeded at 250,000 cells / well in six-well plates coated with poly-D-lysine (1:500). N2A cells were transfected with 3.0 µg of specified plasmid DNA (Lipofectamine LTX; Invitrogen). ™(Waltham, Massachusetts, USA) After overnight transfection, N2A cells were washed twice with 4°C DPBS in pre-chilled centrifuge tubes. N2A cells were then centrifuged at 500xg for 3 minutes at 4°C to form clumps, the supernatant was discarded, and the cells were washed twice with 4°C DPBS. N2A cells were resuspended in 200 µL of lysis buffer (10 mM Tris / Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA; supplemented with a 1:100 protease inhibitor and 1 mM PMSF), and lysed by repeated aspiration 30 times using a 30-G needle. Cell lysates were centrifuged at 20,000 x g for 10 min at 4°C. Soluble postnuclear supernatant (PNS) was transferred to EGFP-TRAP®_MA magnetic beads (Proteintech®, Rosemount, Illinois, USA), which had been equilibrated in dilution / washing buffer (10 mM Tris / Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA, supplemented with 1:100 protease inhibitor and 1 mM PMSF) and incubated at 4°C for 60 min. The EGFP-TRAP®_MA beads were magnetically separated and washed three times with washing buffer. The immune complexes bound to the EGFP-TRAP®_MA beads were dissociated after boiling in 2x protein sample buffer (PSB) at 95°C for 10 min.

[0125] Western blot. EGFP-TRAP®_MA eluent was collected in 2x PBS and subjected to SDS-PAGE gel electrophoresis as previously described [8]. Samples were separated on 13.5% SDS-PAGE and transferred to Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). PVDF membranes were rehydrated in methanol, washed with reverse osmosis water, stained with Ponceau S (P3504, Sigma-Aldrich, St. Louis, USA), and incubated in blocking buffer (2% milk powder (w / v) in 0.1% TBST). PVDF membranes were incubated overnight at 4°C with shaking with 1:2000 rat α-EGFP monoclonal IgG (3H9, Chromotek, Planegg, DE), 1:20000 mouse α-β-actin monoclonal IgG (A2228, Millipore Sigma, Massachusetts, USA), or 1:2000 rabbit α-β-3 tubulin (ab3335, Abcam, Cambridge, UK). The bound primary antibody was recognized by a secondary antibody conjugated to horseradish peroxidase, comprising a 1:10,000 dilution of anti-rat IgG, a 1:20,000 dilution of anti-mouse IgG, or a 1:10,000 dilution of anti-rabbit IgG (Life Technologies, Massachusetts, USA). Secondary antibody imaging was performed on an Azure C600 imaging system (Dublin, California, USA) using a Super Signal™ West Pico PLUS chemiluminescent substrate (34578, Thermo, Illinois, USA) with a 60-second exposure.

[0126] Mass spectrometric evaluation of Rab GTPases associated with LC / A A1 and LC / A3LM. EGFP-TRAP®_MA eluent was collected in clean 2xPBS and separated on gradient (8–16%) stain-free protein gels (5678104, Bio-Rad, Hercules, CA, USA). Samples were separated by electrophoresis in the gels to approximately 1 cm, sufficient to distinguish 250 kDa molecular weight markers. The gels were then fixed in 30% isopropanol and 10% glacial acetic acid for 30 min and washed three times for 10 min each in HPLC-grade water (EM-WX0008-1, VWR, PA, USA). Whole samples were excised from the gels and individually packaged for mass spectrometric analysis at the Taplin Biomass Spectrometry Facility, Harvard Medical School. Mass spectrometry results were received as a compilation list in an Excel spreadsheet. The Excel spreadsheet included Uniprot codes, gene symbols, annotations, molecular weights, and total and unique peptides detected. Samples were analyzed first in an unbiased manner and then in a biased manner. Biased analysis focuses on proteins enriched more than twice that of EGFP, while unbiased analysis includes the entire dataset. The N2A proteomics derived from mass spectrometry used in this study identified over 3500 proteins in total cell lysates, soluble fractions, and / or membrane “precipitates” (supplementary). Figure 4 b).

[0127] Table 3: Protein counts in untreated and nocodaazole-treated Neuro-2a cells identified by mass spectrometry (unbiased and biased). 1 ).

[0128]

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[0178] 49. Gorvel, J.P., et al., rab5 controls early endosome fusion in vitro. Cell, 1991. 64(5): p. 915-25.

[0179] 50. Simon, N.C., K. Aktories and J.T. Barbieri, Novel bacterial ADP-ribosylating toxins: structure and function. Nat Rev Microbiol, 2014. 12(9): p. 599-611.

[0180] 51. Mignogna, M.L. and P. D'Adamo, Critical importance of RAB proteins for synaptic function. Small GTPases, 2018. 9(1-2): p. 145-157.

[0181] 52. Dey, S., G. Banker and K. Ray, Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila. Cell Rep,2017. 18(10): p. 2452-2463.

[0182] 53. Hausser, A. and K. Schlett, Coordination of AMPA receptortrafficking by Rab GTPases. Small GTPases, 2019. 10(6): p. 419-432.

[0183] 54. Saheki, Y. and P. De Camilli, Synaptic vesicle endocytosis. ColdSpring Harb Perspect Biol, 2012. 4(9): p. a005645.

[0184] 55. Szodorai, A., et al., APP anterograde transport requires Rab3A GTPaseactivity for assembly of the transport vesicle. J Neurosci, 2009. 29(46): p.14534-44.

[0185] 56. Schluter, O.M., et al., A complete genetic analysis of neuronal Rab3function. J Neurosci, 2004. 24(29): p. 6629-37.

[0186] 57. Pavlos, N.J., et al., Quantitative analysis of synaptic vesicle Rabsuncovers distinct yet overlapping roles for Rab3a and Rab27b in Ca2+-triggered exocytosis. J Neurosci, 2010. 30(40): p. 13441-53.

[0187] 58. Pavlos, N.J. and R. Jahn, Distinct yet overlapping roles of RabGTPases on synaptic vesicles. Small GTPases, 2011. 2(2): p. 77-81.

[0188] 59. Burns, M.E., et al., Rabphilin-3A: a multifunctional regulator of synaptic vesicle traffic. J Gen Physiol, 1998. 111(2): p. 243-55.

[0189] 60. Fukuda, M., E. Kanno and A. Yamamoto, Rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27A in PC12 cells. J Biol Chem, 2004. 279(13): p. 13065-75.

[0190] 61. Li, T., et al., Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis. Elife, 2022. 11.

[0191] 62. Pirazzini, M., et al., Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology. Pharmacol Rev, 2017. 69(2): p. 200-235.

[0192] 63. Pfeffer, S.R., Rab GTPases: master regulators that establish the secretory and endocytic pathways. Mol Biol Cell, 2017. 28(6): p. 712-715.

[0193] 64. Zuverink, M., et al., A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation. InfectImmun, 2015. 83(7): p. 2714-24.

[0194] 65. Schneider, CA, WS Rasband and KW Eliceiri, NIH Image toImageJ: 25 years of image analysis. Nat Methods, 2012. 9(7): p. 671-5.

[0195] 66. Schindelin, J., et al., Fiji: an open-source platform for biological-image analysis. Nat Methods, 2012. 9(7): p. 676-82.

[0196] Example 2: The N-terminus plays only a minor role in the duration of BoNT / A action.

[0197] introduction

[0198] In our study, we identified two amino acid alterations (Q7P, V14G) near the N-terminus of BoNT / ALC that transformed the punctate (vesicle-associated) intracellular localization of BoNT / A3 into diffuse intracellular localization. This suggests that the BoNT / AN terminus participates in intracellular light chain (LC) migration by mediating synaptic vesicle associations. To further examine this, we constructed a recombinant BoNT / A3 whole toxin containing these two amino acid alterations, purified the whole toxin, and examined the duration of its effect in primary neuron cultures and mice.

[0199] method

[0200] Generation of the recombinant gene for expressing BoNT / A3 Q7P, V14G. Following the manufacturer's instructions (New England Biolabs), the BoNT / A3 gene was amplified by PCR using total genomic DNA isolated from Clostridium botulinum subtype A3 strains (provided courtesy of the CDC) and Fusion HotStart Flex 2x master mix. To introduce a two-amino acid mutation at the N-terminus of the toxin, two nucleotide substitutions were incorporated into the 5' PCR primers: a mutant Gln... 7 To Pro (CAA to CCA) and Val 14 To Gly (GTA to GGA). The bond / A3 mutant gene was amplified using primers A3-Nde-5' and A3-Sal-3'. A3-Nde-5' GG CA TATG CCATTTGTTAATAAACCATTTAATTATAGAGATCCTGGAAATGGTG (SEQ ID NO: 1). A3-SalI-3'GC GTCGAC CTTACAGTGAACTTTCTCCCCATCCATCATC (SEQ ID NO: 2).

[0201] The nucleotide sequence of the mutated recombinant toxin gene was verified by DNA sequencing. The recombinant gene was then inserted into the modular Clostridium expression vectors pMTL82152 and pMTL83152 [1]. The recombinant expression vector was transferred to the non-toxic Clostridium botulinum expression host strain Hall A-hyper / tox via conjugation transfer from the Escherichia coli donor strain CA434. - In addition, the expression of recombinant mutant toxins was evaluated as described above [2].

[0202] Production of BoNT / A3 Q7P, V14G. Using the pMTL82152 vector [1], on the non-toxic Clostridium botulinum strain HallA-hyper / tox - [2] The recombinant BoNT / A3-2M (Q7P, V14G) was expressed.

[0203] rBoNT / A3-2M was purified using the previously described BoNT / A1 purification method [3], with an additional chromatographic step following the DEAE (pH 8.0) column, namely, removal of the complex protein using a 1.5 mL p-aminobenzyl-1-thio-bD-galactopyranoside (pABTG) agarose affinity column as described above [4]. The purified toxin was filtered through a 0.2 mm filter and mixed with an equal volume of 80% sterile glycerol and stored at -20°C.

[0204] Purified BoNT / A3-2M was analyzed by 4-12% Bis-Tris (Novex NuPAGE, Life Technologies) gel electrophoresis (non-reducing or reduced with 100 mM DTT), stained with InstantBlue (Expedeon), and its concentration and purity were determined by spectroscopic methods (absorbance at 278 nm) and density scanning.

[0205] Mouse bioassay. The activity of purified 2M-rA3 was determined using the standard mouse intraperitoneal bioassay (MBA) as described above [3,5,6]. Briefly, BoNT / A3-2M was diluted in gelatin phosphate buffer (0.03M sodium phosphate, pH 6.3, 0.2% gelatin) to final concentrations of 44, 88, 134, 178, 266, and 356 pg / mL. Four female ICR mice (18–22 g) in each group were intraperitoneally injected (ip) with 0.5 mL of each dilution, and observed for 4 days for signs of botulism and death. Specific activity was calculated according to the method of Reed and Muench [7].

[0206] Cell-based assays. Primary rat spinal cord (RSC) cells were prepared from E15 Sprague Dawley rat pups and seeded at a density of 50,000 cells / well into wells pre-coated with 0.01% poly-L-ornithine and 8.3 µg / cm³ of iodine. ² RSC cells were maintained in 96-well TPP plates from Matrigel. Cells were maintained in Neurobasal medium supplemented with B27, glutamax, and penicillin / streptomycin (both from Life Technologies) [8,9] and used for toxin assays after 19 days of culture. Cells were exposed to 15 mouse LD50 units (mLD50) of wild-type (wt) BoNT / A3 or 15 mL D50 of BoNT / A3-Q7P, V14G for 48 hours, followed by washing away all extracellular toxins and incubation in fresh medium, with medium changed twice weekly. Triple replicates of cell samples were collected at specified time points in 1xLDS sample buffer (Life Technologies), and the percentage of SNAP-25 cleaved by BoNT in cell lysates was analyzed using Western blotting and density scanning. A monoclonal anti-SNAP-25 antibody was used to identify both cleaved and uncleaved SNAP-25 (Synaptic Systems, Germany), as previously described [8,9]. Images were acquired using PhosphaGlo reagent (KPL, Gaithersburg, MD) and an Azure C600 imaging system. The percentage of uncut SNAP-25 reflects cell recovery from BoNT poisoning.

[0207] result

[0208] The N-terminus plays only a minor role in the duration of action of BoNT / A3. SDS-PAGE analysis of the purified recombinant BoNT / A3-Q7P,V14G2M showed a single 150 kDa band in the non-reduced sample; in the reduced sample, over 90% of the toxins were observed as 100 kDa and 50 kDa LC and HC bands, respectively, indicating a double-stranded toxin content >90%. Figure 8 ).

[0209] Standard mouse biological assays determined that the specific activity of the purified 2M-rA3 toxin was approximately 1 x 10⁻⁶. 7 LD50 / mg (approximately 100 pg / LD50). For comparison, the specific activity of wild-type BoNT / A3 is approximately 5.8 x 10⁻⁶. 7 LD50 / mg (approximately 17 pg / LD50) [10,11].

[0210] To determine whether the N-terminus affected the duration of action, wild-type BoNT / A3 or BoNT / A3-Q7P, V14G was locally injected into the gastrocnemius muscle of mice, and rotarod tests and toe abduction score (DAS) tests were performed to determine motor neuron deficits and local paralysis, respectively. Although small global motor neuron deficits were detected with both toxins (consistent with our previous data), the highest DAS scores for both toxins reached approximately 4 points. Figure 9 BoNT / A3 and mutant BoNT / A3-Q7P, V14G showed almost no difference in DAS score recovery, indicating that the N-terminal mutation had little effect on the duration of action.

[0211] To further analyze the duration of action, primary rat spinal cord cells were exposed to wild-type BoNT / A3 or BoNT / A3-Q7P, V14G, and recovery from SNAP-25 cleavage was monitored over time. Cells exposed to wild-type BoNT / A3 and BoNT / A3-Q7P, V14G showed almost no difference in SNAP-25 cleavage recovery rates. Figure 10 ).

[0212] in conclusion

[0213] In summary, these data indicate that the N-terminus of BoNT / A3 LC has little or no effect on the duration of action.

[0214] Example 3: Functional BoNT / A LC can be introduced into human neurons via mRNA transfection, thereby producing a persistent effect determined by the LHD domain.

[0215] introduction

[0216] The use of mRNA as a therapeutic agent has been demonstrated in the vaccine field. In this case, mRNA is packaged in lipid nanoparticles and delivered to host cells, which then produce antigens and release them into the circulatory system, thereby generating antibodies against those antigens. However, mRNA can also be used to generate desired proteins within target cells without introducing DNA material into the cells that may interact with the host genome. The challenge of this approach lies in how to successfully deliver mRNA to a sufficient number of target cells to achieve a therapeutic effect. In this example, BoNT-LC mRNA was targeted and delivered to human (iPSC-derived) neurons. Neurons, especially those derived from human iPSCs, are difficult to transfect with either DNA or mRNA, which may hinder the therapeutic use of BoNT-LC mRNA. However, as we demonstrate in this example, we have successfully achieved high transfection efficiency (up to 90%) and observed measurable and durable exocytosis inhibition in human neurons. Since the N-terminus of BoNT / A LC does not appear to determine the duration of action, we employed a novel approach to investigate the effects of LHD by transfecting human induced pluripotent stem cell (hiPSC)-derived motor neurons (provided by BrainXCell) with a synthetic mRNA construct encoding BoNT / A LC and its derivatives. Several hurdles had to be overcome for this experiment. We had to create an mRNA construct capable of evading intracellular cellular immune defenses to express functionally and correctly folded LC intracellularly, and we had to design an optimized cell transfection protocol for hiPSC-derived neurons, which are notoriously difficult to transfect. Initial attempts to use lab-prepared mRNAs with standard nucleotide modifications and capping methods resulted in low transfection efficiencies (15-30%), even after optimization for neuronal transfection.

[0217] method

[0218] Neuronal cell culture. Spinal motor neurons derived from human induced pluripotent stem cells (hiPSCs) were supplied by BrainXell (Madison, Wisconsin) and used substantially as described previously

[12] . Cells were seeded at a density of approximately 38,000 cells / well in 96-well TPP plates (Techno Plastic Products, Midwest Scientific, Valley Garden, Missouri) pre-coated with 0.01% poly-L-ornithine and 8.3 µg / cm ²Matrigel (BD Biosciences, East Rutherford, NJ) was used, and cells were maintained according to the manufacturer's instructions. Recommended culture media were used, consisting of equal portions of Neurobasal and DMEM / F12 medium supplemented with B27, N2, and GlutaMAX (Life Technologies). Cells were cultured for 3 days prior to mRNA transfection and for 7 days prior to LC / A1 mRNA transfection in LNP formulation.

[0219] All mRNA constructs were purchased from RiboPro (Netherlands) and manufactured according to the following modifications: RNA synthesis de novo Template / Plasmid Reduced dsRNA content Upgrade to Cap1 150 nt Poly-A Tail Buffer: RNase-free water Concentration: 1 μg / μL The production status of the lipid formulation BoNT / A1 LC mRNA is as follows: RNA synthesis de novo Template / Plasmid Reduced dsRNA content Upgrade to Cap1 150 nt Poly-A Tail ciLNP prepared in PBS Concentration: 100 ng / μL The following constructs were created: LC / A1 aa1-450, LC / A3 aa1-446, LC / A3-Q7P, V14G aa1-446, LC / A1 (A3LHD) A1aa1-267 – A3 a268-357 – A1aa358-450, LC / A3 (A1LHD)A3aa1-267 – A1aa268-357-A3aa358-446. The amino acid and nucleotide sequences are provided below.

[0220] Lipofectamine MessengerMax transfection was performed using Lipofectamine MessengerMax reagent (Invitrogen) in OptiMEM serum-depleted medium (Gibco). The Lipofectamine complex was prepared by diluting the Lipofectamine MessengerMax reagent in OptiMEM medium at a volume ratio of 3:100. The mixture was incubated at room temperature for 10 minutes. Customized LC / A1 and A3 mRNA constructs were diluted to a concentration of 40 ng / µL in OptiMEM medium. Equal volumes of Lipofectamine MessengerMax solution and mRNA dilution were combined and incubated at room temperature for 5 minutes. 10 μL of the complex was added to each well, resulting in a final volume of 0.15 μL of Lipofectamine MessengerMax reagent and 200 ng of mRNA. Cells were incubated at 37°C and 5% CO2 for 24 hours before removing the complex and washing the cells with culture medium. Starting from day 6 post-transfection, cell samples (n=5) were collected approximately every 10 days, placed in 1x NuPAGE LDS sample buffer, and heated at 95°C for 10 minutes. Samples were stored at -20°C. C is used for Western blot analysis.

[0221] RiboPro LNP transfection. Seven days after seeding hiPSC-derived motor neurons, cells were transfected using RiboPro's lipid nanoparticle formulation containing LC / A1 mRNA. 100 or 200 ng of LC / A1 mRNA per well, encapsulated in LNPs, was added directly to cells or diluted in OptiMEM medium before addition. Cells and the complex were incubated at 37°C and 5% CO2, and samples were collected in 1 x NuPAGE LDS sample buffer after 24 and 42 hours. Samples were heated at 95°C for 10 min and stored at -20°C for Western blot analysis.

[0222] Western blot analysis, as previously described, was performed using Western blotting and density scanning to analyze the percentage of cleaved SNAP-25 in cell lysates. A monoclonal anti-SNAP-25 antibody was used to simultaneously identify cleaved and uncleaved SNAP-25 (Synaptic Systems, Germany) ([8,9]). Images were acquired using PhosphaGlo reagents (KPL, Gaithersburg, MD) and an Azure C600 imaging system. Density scanning was performed using Azure Spot software version 2.0.062, and a graph of cleaved SNAP-25 (percentage compared to uncleaved) over time was created in Excel using the mean and standard deviation of five replicates. Data points were fitted with a logarithmic trend line.

[0223] result

[0224] BoNT / A LC mRNA transfection of neurons produces active intracellular LC, the duration of which is determined by LHD. Transfection of motor neurons with GFP control mRNA showed approximately 70% cell transfection. For the BoNT / A LC construct, transfection efficiency was assessed by the percentage of SNAP-25 cleavage at the first time point on day 6 post-transfection. At this time point, the percentage of SNAP-25 cleavage ranged from 11% to over 60%, with BoNT / A1 LC producing an average SNAP-25 cleavage of 40 ± 12%. Figure 11 This indicates extremely high transfection efficiency, and the lower values ​​in some constructs are associated with the previously identified lower LC potency of the whole toxin (see Example 2). The decrease in the percentage of cleaved SNAP-25 was monitored at intervals of approximately 10–14 days over a period up to 60 days post-transfection. A logarithmic decrease in the percentage of cleaved SNAP-25 was observed for all LC constructs, with the most rapid decrease observed in LC / A3-Q7P, V14G, and LC / A1 (A3LHD). A similar rapid rate of decrease in LC activity was also observed in LC / A3, although this LC, due to its higher potency compared to LC / A3-Q7P, V14G, had a higher initial cleavage rate, thus resulting in cleaved SNAP-25 observed over a longer total time period. This is consistent with the known dose-dependent duration of action effect of BoNT. LC / A1 and LC / A3 (A1LHD) showed a much slower rate of decrease in cleaved SNAP-25, indicating that LC / A1 and LC / A3 containing A1 LHD exhibit longer duration of LC activity in motor neurons.

[0225] The RiboPro LNP formulation of LC / A1 mRNA achieved near-complete transfection efficiency in human motor neurons. To examine whether the lipid formulation provided by RiboPro could further improve transfection efficiency, hiPSC-derived motor neurons were transfected with the LC / A1 lipid formulation, and the percentage of SNAP-25 cleavage in the cell lysate was analyzed at 24 or 42 hours. Depending on the amount of mRNA used and the incubation time with cells, a maximum of approximately 85% SNAP-25 cleavage was achieved, indicating a transfection efficiency greater than 80%. Figure 12 ).

[0226] in conclusion

[0227] These data demonstrate successful transfection of human neurons with BoNT / A LC, resulting in functionally active LCs within the targeted neurons. The duration of LC activity is long, consistent with the activity observed in hiPSC-derived neurons poisoned by BoNT / A protein. The data further indicate that the low homology domain (LHD) determines the long duration of action of BoNT / A LC. Finally, we demonstrate that even cells notoriously difficult to transfect, such as hiPSC-derived neurons, can be transfected very efficiently using mRNA formulated as nanoparticles. In summary, these data suggest that utilizing BoNTLC mRNA constructs as an alternative to protein therapy is therapeutically feasible. Furthermore, the duration of action can be customized by sequence modification of the mRNA within the MLD domain. Since nanoparticle targeting is not limited to neurons, other targets, including inflammatory cells or cancer cells, are also viable targets for this type of therapy.

[0228] Amino acid and nucleotide sequences of the mRNA construct: WT LC / A1 Amino acid sequence (SEQ ID NO: 3): MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL- DNA sequence (SEQ ID NO:4):

[0229] WT LC / A3

[0230] Amino acid sequence (SEQ ID NO: 5): MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSRDAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLTRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL- DNA sequence (SEQ ID NO: 6):

[0231] JV LC / A3

[0232] Amino acid sequence (SEQ ID NO: 7): MPFVNKPFNYRDPGNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSRDAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLTRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL- DNA sequence (SEQ ID NO: 8):

[0233] LC / A1 (A3 LHD)

[0234] Amino acid sequence (SEQ ID NO: 9): MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSRDAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLTRGFTELEFVNPFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL- DNA sequence (SEQ ID NO: 10): [[ID=IO]]

[0235] LC / A3 (A1 LHD)

[0236] Amino acid sequence (SEQ ID NO: 11): MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL- DNA sequence (SEQ ID NO:12):

[0237] References for Examples 2 and 3

[0238] 1. Heap, J.T.; Pennington, O.J.; Cartman, S.T.; Minton, N.P. Amodular system for Clostridium shuttle plasmids. J Microbiol Methods 2009,78, 79 - 85, doi:S0167 - 7012(09)00129 - 8 [pii] 10.1016 / j.mimet.2009.05.004 [doi].

[0239] 2. Bradshaw, M.; Tepp, W.H.; Whitemarsh, R.C.; Pellett, S.; Johnson,E.A. Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from aNontoxigenic Clostridium botulinum Expression System. Appl Environ Microbiol2014, 80, 7415 - 7422, doi:10.1128 / aem.01795 - 14.

[0240] 3. Malizio, C.J.; Goodnough, M.C.; Johnson, E.A. Purification ofClostridium botulinum type A neurotoxin. Methods in molecular biology(Clifton, N.J.) 2000, 145, 27 - 39, doi:10.1385 / 1 - 59259 - 052 - 7:27.

[0241] 4. Moberg, L.J.; Sugiyama, H. Affinity chromatography purification oftype A botulinum neurotoxin from crystalline toxic complex. Appl EnvironMicrobiol 除了对专利文本进行翻译,我还可以为你提供专业的翻译建议和技巧,帮助你更好地理解和处理专利文本的翻译工作。如果你有任何关于专利文本翻译的问题,随时都可以问我。1978, 35, 878 - 880.

[0242] 5. Hatheway, C.L. Botulism. In Laboratory diagnosis of infectiousdiseases: principles and practice., Balows, A., Hausler, W.H., Ohashi, M.,Turano, M.A., Eds. Springer-Verlag: New York, 1988; Vol. 1, pp. 111–133.

[0243] 6. Schantz, E.J.; Kautter, D.A. Standardized assay for Clostridiumbotulinum toxins. Journal - Association of Official Analytical Chemists 1978,61, 96-99.

[0244] 7. Reed, L.; Muench, H. A simple method of estimating fifty percentendpoints. Am. J. Hyg. 1938, 27, 493-497.

[0245] 8. Pellett, S.; Tepp, W.H.; Clancy, C.M.; Borodic, G.E.; Johnson,E.A. A neuronal cell-based botulinum neurotoxin assay for highly sensitiveand specific detection of neutralizing serum antibodies. FEBS letters 2007,581, 4803-4808, doi:10.1016 / j.febslet.2007.08.078.

[0246] 9. Pellett, S.; Tepp, W.H.; Toth, S.I.; Johnson, E.A. Comparison ofthe primary rat spinal cord cell (RSC) assay and the mouse bioassay forbotulinum neurotoxin type A potency determination. Journal of pharmacologicaland toxicological methods 2010, 61, 304-310, doi:10.1016 / j.vascn.2010.01.003.

[0247] 10. Tepp, W.H.; Lin, G.; Johnson, E.A. Purification andcharacterization of a novel subtype a3 botulinum neurotoxin. Appl EnvironMicrobiol 2012, 78, 3108-3113, doi:10.1128 / aem.07967-11.

[0248] 11. Gardner, A.; Tepp, W.H.; Bradshaw, M.; Barbieri, J.T.; Pellett,S. Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light ChainLocalization to the Intracellular Plasma Membrane. International journal ofmolecular sciences 2021, 22, doi:10.3390 / ijms222011115.

[0249] 12. Whitemarsh, R.C.; Strathman, M.J.; Chase, L.G.; Stankewicz, C.;Tepp, W.H.; Johnson, E.A.; Pellett, S. Novel application of human neuronsderived from induced pluripotent stem cells for highly sensitive botulinumneurotoxin detection. Toxicological sciences : an official journal of theSociety of Toxicology 2012, 126, 426-435, doi:10.1093 / toxsci / kfr354.

[0250] Other sequences – mRNA sequences

[0251] >WT LC / A1 (SEQ ID NO: 39)

[0252]

[0253] >WT LC / A3 (SEQ ID NO: 40)

[0254]

[0255] >JV LC / A3 (SEQ ID NO: 41)

[0256]

[0257] >LC / A1 (A3 LHD) (SEQ ID NO: 42)

[0258]

[0259] >LC / A3 (A1 LHD) (SEQ ID NO: 43)

[0260]

[0261] Example 4

[0262] Since our mRNA studies have shown that, in addition to LHD, the N-terminus can also affect the duration of action, and we have already identified MLD, we further investigated the effects of MLD and the N-terminus (A3-Q7P, V14G) in hiPSC-derived motor neuron mRNA assays. Because RiboPro nanoparticles offer better conversion efficiency, we used these nanoparticles for mRNA delivery in our experiments.

[0263] method

[0264] Neuronal cell culture. Spinal motor neurons derived from human induced pluripotent stem cells (hiPSCs) were supplied by BrainXell (Madison, Wisconsin) and used substantially as described previously

[12] . Cells were seeded at a density of approximately 38,000 cells / well in 96-well TPP plates (Techno Plastic Products, Midwest Scientific, Valley Garden, Missouri) pre-coated with 0.01% poly-L-ornithine and 8.3 µg / cm 2 Matrigel (BD Biosciences, East Rutherford, NJ) was used, and maintenance was performed according to the manufacturer's instructions. Recommended culture media containing equal portions of Neurobasal and DMEM / F12 medium, supplemented with B27, N2, and GlutaMAX (Life Technologies) were used. Cells were cultured for 3 days prior to mRNA transfection and for 7 days prior to LC / A1 mRNA transfection with the LNP formulation.

[0265] mRNA constructs. All mRNA constructs were purchased from RiboPro (Netherlands) and manufactured according to the following modifications: RNA synthesis de novo Template / Plasmid Reduced dsRNA content Upgrade to Cap1 150 nt Poly-A Tail Buffer: RNase-free water Concentration: 1 μg / μL The production status of the lipid formulation BoNT / A1 LC mRNA is as follows: RNA synthesis de novo Template / Plasmid Reduced dsRNA content Upgrade to Cap1 150 nt Poly-A Tail ciLNP prepared in PBS Concentration: 100 ng / μL The following constructs were created: LC / A1 aa1-450, LC / A3 aa1-446, LC / A3-Q7P, V14G aa1-446, LC / A1 (A3MLD) A1aa1-295 – A3 a296-357 – A1aa358-450, LC / A3 (A1MLD)A3aa1-295 – A1aa296-357-A3aa358-446, and LC / A3-Q7P, V14G(A1MLD) A3aa1-267 –A1aa296-357-A3aa358-446. The amino acid and nucleotide sequences are provided below.

[0266] RiboPro LNP transfection. Seven days after seeding hiPSC-derived motor neurons, cells were transfected using RiboPro's lipid nanoparticle formulation. 100 or 200 ng LC / A1 mRNA / well containing LNPs was added directly to cells, or diluted in OptiMEM medium before addition. Cells and the complex were incubated at 37°C and 5% CO2, and samples were collected in 1x NuPAGE LDS sample buffer after 3, 6, 9, 15, 21, 30, 42, and 56 days. Samples were heated at 95°C for 10 minutes and stored at -20°C for Western blot analysis. Note: The LC / A1 mRNA in the nanoparticle formulation was expired; therefore, it was transfected using the RNAMax transfection reagent described above.

[0267] Western blot analysis. As previously described, the percentage of cleaved SNAP-25 in cell lysates was analyzed using Western blot and density scanning, with a monoclonal anti-SNAP-25 antibody that simultaneously identifies cleaved and uncleaved SNAP-25 (Synaptic Systems, Germany) ([8,9]). Images were acquired using PhosphaGlo reagents (KPL, Gaithersburg, MD) and an Azure C600 imaging system. Density scanning was performed using Azure Spot software version 2.0.062, and graphs of cleaved SNAP-25 (percentage compared to uncleaved) over time were created in Excel using the mean and standard deviation of five replicates. Data points were fitted with logarithmic trend lines.

[0268] result

[0269] BoNT / A LC mRNA transfection in neurons produces functional intracellular LCs in all or almost all cells, with the duration of action determined by the N-terminus and MLD. Transfection of motor neurons with GFP control mRNA showed nearly 100% cell transfection. For the BoNT / A LC construct, transfection efficiency was assessed by the percentage of SNAP-25 cleavage at the first time point on day 6 post-transfection. At this time point, the percentage of SNAP-25 cleavage ranged from 77% to 100%, except for BoNT / A1 LC transfected using RNAMax reagent, which produced a SNAP-25 cleavage rate of 37%. Figure 14 The lower values ​​for some constructs were associated with previously identified lower LC potency of the whole toxin (see Example 2). This indicates extremely high transfection efficiency sufficient for therapeutic use. The decrease in the percentage of SNAP-25 cleavage was monitored over a period of up to 56 days post-transfection.

[0270] Except for LC / A1, all LC constructs showed a logarithmic decrease in the percentage of SNAP-25 cleavage. Cells treated with LC / A1 maintained a constant level of SNAP-25 cleavage throughout the test period. The most rapid decreases in SNAP-25 cleavage were observed in LC / A3-Q7P,V14G, LC / A3-Q7P,V14G-A1MLD, and LC / A1-A3MLD. This indicates that the Q7P,V14G mutation at the LC / AN terminus dominates in determining the short duration of action, and that A3-MLD similarly determines a shorter duration of action. LC / A3wt and LC / A3wt-A1MLD resulted in similar but slower decreases in SNAP-25 cleavage. This indicates that although A1MLD can guide the membrane localization of LC / A3, it is insufficient to establish a longer duration of action for LC / A3 (Gardner AP, Barbieri JT, Pellett S. How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane. Toxins (Basel). 22 Nov 2022;14(12):814. doi: 10.3390 / toxins14120814. PMID: 36548711; PMCID: PMC9783275). Overall, the data are consistent with the mRNA data of the LHD mutant, except that A1-MLD did not prolong the duration of action, while A1-LHD appears to have the potential to slightly increase the duration of action of LC / A3 (). Figure 11 ).

[0271] discuss

[0272] These data demonstrate that efficient transfection of human neurons with BoNT / A LC mRNA resulted in the generation of functionally active LCs within the targeted neurons. The duration of LC activity was long, consistent with the activity observed in hiPSC-derived neurons poisoned by BoNT / A protein. The data further indicate that both the N-terminus and MLD domains play a role in regulating the duration of action of BoNT / A LCs. In summary, these data suggest that utilizing BoNT LC mRNA constructs as an alternative to protein therapy is therapeutically feasible. Furthermore, the duration of action can be tailored by sequence modifications of the mRNA in the N-terminal and MLD domains. Since nanoparticle targeting is not limited to neurons, other targets, including inflammatory cells or cancer cells, are also viable targets for this type of therapy. Additionally, neuronal subpopulations, such as pain-transmitting neurons, may also be targets for this technology.

[0273] based on Figure 14 The data show that A1-N-Term and A1-MLD jointly contribute to the long-lasting effect of BoNT / A1. These data support the role of the N-terminus (N-terminus) and MLD (LHD) of LC / A1 in inducing a two-step mechanism: the N-terminus (A1-N-terminal residues 1-17 of A1) mediates LC / A1 transport to the plasma membrane via Rab-dependent vesicles, followed by A1-MLD (residues 275-334 of A1) mediating a stable association with the plasma membrane; these two steps together induce the high potency and long-lasting effect of BoNT / A1. Based on these findings and the fact that the three-dimensional structures of the light chains of the seven BoNT serotypes are conserved, chimeras can be constructed to modulate the potency and duration of action of BoNT.

[0274] Sequence of Example 4

[0275] >WT LC / A1 (SEQ ID NO: 44)

[0276] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIV RGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKF DKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL-

[0277] DNA seq (SEQ ID NO: 45)

[0278]

[0279] >WT LC / A1 (A3 MLD) (SEQ ID NO: 46)

[0280] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLWQKKFSRDAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL-

[0281] DNA seq (SEQ ID NO: 47)

[0282]

[0283] >JV LC / A3 (SEQ ID NO: 48)

[0284] MPFVNKPFNYRDPGNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSRDAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLTRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0285] DNA seq (SEQ ID NO: 49)

[0286]

[0287] >JV LC / A3 (A1 MLD) (SEQ ID NO: 50)

[0288] MPFVNKPFNYRDPGNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKAAFKEFYRVLTRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0289] DNA Seq (SEQ ID NO: 51)

[0290]

[0291] >LM LC / A3 (SEQ ID NO: 52)

[0292] MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSRDAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLTRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0293] DNA Seq (SEQ ID NO: 53)

[0294]

[0295] >LM LC / A3 (A1MLD) (SEQ ID NO: 54)

[0296] MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKAAFKEFYRVLTRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0297] DNA Seq (SEQ ID NO: 55)

[0298]

[0299] Other sequences

[0300] 1. BoNT / E

[0301] a. Amino acid sequence LC / E 1-426 (SEQ ID NO: 13)

[0302] 1 MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS

[0303] 61 LKNGDSSYYD PNYLQSDEEK DRFLKIVTKI FNRINNNLSG GILLEELSKA NPYLGNDNTP

[0304] 121 DNQFHIGDAS AVEIKFSNGS QDILLPNVII MGAEPDLFET NSSNISLRNN YMPSNHGFGS

[0305] 181 IAIVTFSPEY SFRFNDNSMN EFIQDPALTL MHELIHSLHG LYGAKGITTK YTITQKQNPL

[0306] 241 ITNIRGTNIE EFLTFGGTDL NIITSAQSND IYTNLLADYK KIASKLSKVQ VSNPLLNPYK

[0307] 301 DVFEAKYGLD KDASGIYSVN INKFNDIFKK LYSFTEFDLA TKFQVKCRQT YIGQYKYFKL

[0308] 361 SNLLNDSIYN ISEGYNINNL KVNFRGQNAN LNPRIITPIT GRGLVKKIIR FC

[0309] a. Amino acid sequence BoNT / E 1-1251 (SEQ ID NO: 14)

[0310] 1 MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS

[0311] 61 LKNGDSSYYD PNYLQSDEEK DRFLKIVTKI FNRINNNLSG GILLEELSKA NPYLGNDNTP

[0312] 121 DNQFHIGDAS AVEIKFSNGS QDILLPNVII MGAEPDLFET NSSNISLRNN YMPSNHRFGS

[0313] 181 IAIVTFSPEY SFRFNDNCMN EFIQDPALTL MHELIHSLHG LYGAKGITTK YTITQKQNPL

[0314] 241 ITNIRGTNIE EFLTFGGTDL NIITSAQSND IYTNLLADYK KIASKLSKVQ VSNPLLNPYK

[0315] 301 DVFEAKYGLD KDASGIYSVN INKFNDIFKK LYSFTEFDLR TKFQVKCRQT YIGQYKYFKL

[0316] 361 SNLLNDSIYN ISEGYNINNL KVNFRGQNAN LNPRIITPIT GRGLVKKIIR FCKNIVSVKG

[0317] 421 IRKSICIEIN NGELFFVASE NSYNDDNINT PKEIDDTVTS NNNYENDLDQ VILNFNSESA

[0318] 481 PGLSDEKLNL TIQNDAYIPK YDSNGTSDIE QHDVNELNVF FYLDAQKVPE GENNVNLTSS

[0319] 541 IDTALLEQPK IYTFFSSEFI NNVNKPVQAA LFVSWIQQVL VDFTTEANQK STVDKIADIS

[0320] 601 IVVPYIGLAL NIGNEAQKGN FKDALELLGA GILLEFEPEL LIPTILVFTI KSFLGSSDNK

[0321] 661 NKVIKAINNA LKERDEKWKE VYSFIVSNWM TKINTQFNKR KEQMYQALQN QVNAIKTIIE

[0322] 721 SKYNSYTLEE KNELTNKYDI KQIENELNQK VSIAMNNIDR FLTESSISYL MKIINEVKIN

[0323] 781 KLREYDENVK TYLLNYIIQH GSILGESQQE LNSMVTDTLN NSIPFKLSSY TDDKILISYF

[0324] 841 NKFFKRIKSS SVLNMRYKND KYVDTSGYDS NININGDVYK YPTNKNQFGI YNDKLSEVNI

[0325] 901 SQNDYIIYDN KYKNFSISFW VRIPNYDNKI VNVNNEYTII NCMRDNNSGW KVSLNHNEII

[0326] 961 WTFEDNRGIN QKLAFNYGNA NGISDYINKW IFVTITNDRL GDSKLYINGN LIDQKSILNL[[ID=十六]] [[ID=十七]]

[0327] 1021 GNIHVSDNIL FKIVNCSYTR YIGIRYFNIF DKELDETEIQ TLYSNEPNTNILKDFWGNYL

[0328] 1081 LYDKEYYLLN VLKPNNFIDR RKDSTLSINN IRSTILLANR LYSGIKVKIQRVNNSSTNDN

[0329] 1141 LVRKNDQVYI NFVASKTHLF PLYADTATTN KEKTIKISSS GNRFNQVVVMNSVGNCTMNF

[0330] 1201 KNNNGNNIGL LGFKADTVVA STWYYTHMRD HTNSNGCFWN FISEEHGWQE K

[0331] b. DNA sequence BoNT / E (SEQ ID NO: 15)

[0332] It should be noted that some of the tags in the original text seem to be in an incorrect format or might be special codes with unclear meanings. This translation attempts to maintain their integrity as much as possible while converting the text part to English. If there are specific requirements or corrections regarding these tags, it would be beneficial for a more accurate translation.1 atgccaaaaa ttaatagtttt taattataat gatcctgtta atgatagaac attttatat

[0333] 61 attack gcggttgtca agaattttat aaatcattta atattatgaa aaatattgg

[0334] 121 atattccag agagaatgt aattggtaca accccaag atttcatcc gcctactca

[0335] 181 ttaaaaatg gagatagtag ttattatgac cctattatt tacaagtga tgagaaag

[0336] 241 gatagatttt taaaaatagt cacaaaaata tttaatagaa taataata tctttcagga

[0337] 301 gggatttta taggaact gtcaaagct aatccatatt taggaatga taatactcca

[0338] 361 gatacaat tccatattgg tgatgcatca gcagttgaga ttaattctc aaatggtagc

[0339] 421 ciegacatac tattacta tgttatta atgggagcag agcctgattt atttgaact

[0340] 481 aacagttcca atatttctct aagaaataat tatatgccaa gcaatcaccg ttttggatca

[0341] 541 atagctatag taacattctc acctgaat tcttttagat ttaatgata ttgtatgaat

[0342] 601 gatttattc aagatcctgc tcttacatta atgcatgaat taatacattc attacatgga

[0343] 661 ctatatgggg ctaagggat tactacaaag tatactata cacaaaaaaatccccta

[0344] 721 atachaata tagaggtac aaatattgaa gatttcttta cttttggagg tactgattta

[0345] 781 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc tgattataaa

[0346] 841 aaatagcgt ctaaacttag caagtacaa gtactaatc cactacttaa tccttataaa

[0347] 901 gatgttttg aagcaagta tggattagat aagatgcta gcggaattta ttcggtaaat

[0348] 961 aaaaaaaat ttaatgatat ttttaaaaaa tattacagct tacggaatt tgatttacga

[0349] 1021 actaaatttc aagttaaatg taggcaact tattggac agtataataacttcaactt

[0350] 1081 tcaacttgt taaatgattc tatttataat atcagaag gctataataataattta

[0351] 1141 aggtaatt tagaggaca gatgcaat ttaatccta gatttattacaccattaca

[0352] 1201 ggtagaggac tagtaaaaa aatcattaga ttttgtaaaa atattgtttctgtaaaaggc

[0353] 1261 ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgtggcttccgag

[0354] 1321 aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatacagtaacttca

[0355] 1381 aataataatt atgaaaatga tttagatcag gttattttaa atttaatagtgaatcagca

[0356] 1441 ctggacttt cagatgaaaa attaattta actatccaaa atgatgcttatataccaaaa

[0357] 1501 tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaacttaatgtattt

[0358] 1561 ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatctcacctcttca

[0359] 1621 attgatacag cattattaga acaacctaaa atatatacat ttttttcatcagaatttatt

[0360] 1681 aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggatacaaaagtgtta

[0361] 1741 gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgcagatatttct

[0362] 1801 atagttgttc catatatagg tcttgcttta aatataggaa atgaagcacaaaaaggaaat

[0363] 1861 tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttgaacccgagctt

[0364] 1921 ttaattccta caattttagt attcacgata aaatcttttt taggttcatctgataataaa

[0365] 1981 aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaaatggaaagaa

[0366] 2041 gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatttaataaaaga

[0367] 2101 aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg caattaaaacaataatagaa

[0368] 2161 tctaagtata atagttatac tttagaggaa aaaaatgagc ttacaaataaatatgatatt

[0369] 2221 aagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataatatagacagg

[0370] 2281 ttcttaactg aaagttctat atcctattta atgaaaataa taaatgaagtaaaaattaat

[0371] 2341 aaattaagag aatatgatga gaatgtcaaa acgtattat tgaattatattatacaacat

[0372] 2401 ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaactgataccctaaat

[0373] 2461 aatagtattc cttttaagct ttcttcttat acagatgata aaattttaatttcatatttt

[0374] 2521 aataaattct ttaagagaat taaaagtagt tcagttttaa atatgagatataaaaaatgat

[0375] 2581 aaatacgtag atacttcagg atatgattca aatataaata ttaatggagatgtatataaa

[0376] 2641 tatccaacta ataaaaatca atttggaata tataatgata aacttagtgaagttaatata

[0377] 2701 tctcaaaatg attacattat atatgataat aaatataaaa attttagtattagttttgg

[0378] 2761 gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaatacactataata

[0379] 2821 aattgtatga gagataataa ttcaggatgg aaagtatctc ttaatcataatgaaataatt

[0380] 2881 tggacattcg aagataatcg aggaattaat caaaaattag catttaactatggtaacgca

[0381] 2941 aatggtattt ctgattatat aaataagtgg attttgtaa ctataactaatgatagatta

[0382] 3001 ggattcta acctttatat tatggaat ttaatagatc aaaatcaatttaattta

[0383] 3061 ggtatattc atgttagtga caatattatta ttaaatag ttaattgtagttacaga

[0384] 3121 tattattggta tagattttt taatatttt gatataagaat tagagaacagaattcha

[0385] 3181 actttatata gcaatgaacc taatacaat atttgaag atttttgggaattttg

[0386] 3241 ctttatgaca aagatacta tttaat at gtgttaaaac CAatactttattgatagg

[0387] 3301 agaaagatt ctactttaag cattaataat atagaagca ctattctttagctaataga

[0388] 3361 ttatatagtg gaataaagt taaaataca agagttaata atagtagtactaacgaat

[0389] 3421 cttgttagaa agaatgatca gttatatatt aattgtag ccagcaaactcacttattt

[0390] 3481 ccattatatg ctgatacagc taccacaat aaagaaaaaaaaatcatcatct

[0391] 3541 ggcaatagat ttaatcaagt agtagttatg attcagtag gaattgtacaatgaatttt

[0392] 3601 aaaaataata atggaaataa tattgggttg ttaggtttca aggcagatactgtcgttgct

[0393] 3661 agtacttggt attatacaca tatgagagat catacaaaca gcaatggatgtttttggaac

[0394] 3721 tttatttctg aagaacatgg atggcaagaa aaataa

[0395] c. LC / E coordinates, derived from crystal structures PDB: 1T3A and 3FFZ N - terminus 1 - 17 MLD - R1 264 - 287 MLD - R2 288 - 319 MLD - R1 - R2 264 - 319 LHD - R1 - R2 - R3 264 - 343 LC / E1 1 - 412 BoNT / E 1251 2. BoNT / B a. Amino acid sequence LC / B 1 - 437 (SEQ ID NO: 16) 1 MPVTINNFNY NDPIDNNNII MMEPPFARGT GRYYKAFKIT DRIWIIPERY TFGYKPEDFN 61 KSSGIFNRDV CEYYDPDYLN TNDKKNIFLQ TMIKLFNRIK SKPLGEKLLE MIINGIPYLG 121 DRRVPLEEFN TNIASVTVNK LISNPGEVER KKGIFANLII FGPGPVLNEN ETIDIGIQNH 181 FASREGFGGI MQMKFCPEYV SVFNNVQENK GASIFNRRGY FSDPALILMH ELIHVLHGLY 241 GIKVDDLPIV PNEKKFFMQS TDAIQAEELY TFGGQDPSII TPSTDKSIYD KVLQNFRGIV 301 DRNLKVLVCI SDPNININY KNKFKDKYKF VEDSEGKYSI DVESFDKLYK SLMFGFTETN 361 421 YEEISKEHLA VYKIQMC a. Amino acid sequence BoNT / B 1-1291 (SEQ ID NO: 17) 1 MPVTINNFNY NDPIDNNII MMEPPFARGT GRYYKAFKIT DRIWIIPERY TFGYKPEDFN 61 KSSGIFNRDV CEYYDPDYLN TNDKKNIFLQ TMIKLFNRIK SKPLGEKLLE MIINGIPYLG 121 DRRVPLEEFN TNIASVTVNK LISNPGEVER KKGIFANLII FGPGPVLNEN ETIDIGIQNH 181 FASREGFGGI MQMKFCPEYV SVFNNVQENK GASIFNRRGY FSDPALILMH ELIHVLHGLY 241 GIKVDDLPIV PNEKKFFMQS TDAIQAEELY TFGGQDPSII TPSTDKSIYD KVLQNFRGIV 301 DRNLKVLVCI SDPNININY KNKFKDKYKF VEDSEGKYSI DVESFDKLYK SLMFGFTETN 361 421 YEEISKEHLA VYKIQMCKSV KAPGICIDVD NEDLFFIADK NSFSDDLSKN ERIEYNTQSN 481 YIENDFPINE LILDTDLISK IELPSENTES LTDFNVDVPV YEKQPAIKKI FTDENTIFQY 541 LYSQTFPLDI RDISLTSSFD DALLFSNKVY SFFSMDYIKT ANKVVEAGLF AGWVKQIVND 601 FVIEANKSNT MDKIADISLI VPYIGLALNV GNETAKGNFE NAFEIAGASI LLEFIPELLI 661 PVVGAFLLES YIDNKNKIIK TIDNALTKRN EKWSDMYGLI VAQWLSTVNT QFYTIKEGMY 721 KALNYQAQAL EEIIKYRYNI YSEKEKSNIN IDFNDINSKL NEGINQAIDN INNFINGCSV 781 SYLMKKMIPL AVEKLLDFDN ALKKNLLNYI DENKLYLIGS AEYEKSKVNK YLKTIMPFDL 841 SIYTNDTILI EMFNKYNSEI LNNIILNLRY KDNNLIDLSG YGAKVEVYDG VELNDKNQFK 901 LTSSANSKIR VTQNQNIIFN SVFLDFSVSF WIRIPKYKND GIQNYIHNEY TIINCMKNNS 961 GWKISIRGNR IIWTLIDING KTKSVFFEYN IREDISEYIN RWFFVTITNN LNNAKIYING 1021 KLESNTDIKD IREVIANGEI IFKLDGDIDR TQFIWMKYFS IFNTELSQSNIEERYKIQSY 1081 SEYLKDFWGN PLMYNKEYYM FNAGNKNSYI KLKKDSPVGE ILTRSKYNQNSKYINYRDLY 1141 IGEKFIIRRK SNSQSINDDI VRKEDYIYLD FFNLNQEWRV YTYKYFKKEEKLFLAPISD 1201 SDEFYNTIQI KEYDEQPTYS CQLLFKKDEE STDEIGLIGI HRFYESGIVFEEYKDYFCIS 1261 KWYLKEVKRK PYNLKLGCNW QFIPKDEGWT E b. DNA sequence BoNT / B (SEQ ID NO: 18) 1 atgccagtta caataaataa ttttaattat aatgatccta ttgataataa taatattatt 61 atgatggagc ctccatttgc gagaggtacg gggagatatt ataaagcttt taaaatcaca 121 gatcgtattt ggataatacc ggaaagatat acttttggat ataaacctga ggattttaat 181 aaaagttccg gtatttttaa tagagatgtt tgtgaatatt atgatccaga ttacttaaat 241 actaatgata aaaagaatat atttttacaa acaatgatca agttatttaa tagaatcaaa 301 tcaaaaccat tgggtgaaaa gttattagag atgattataa atggtatacc ttatcttgga 361 gatagacgtg ttccactcga agagtttaac acaaacattg ctagtgtaac tgttaataaa 421 ttaatcagta atccaggaga agtggagcga aaaaaaggta ttttcgcaaa tttaataata 481 tttggacctg ggccagtttt aaatgaaaat gagactatag atataggtat acaaaatcat 541 tttgcatcaa gggaaggctt cgggggtata atgcaaatga agttttgccc agaatatgta 601 agcgtattta father agaaaacaaa ggcgcaagta father acgtggatat 661 ttttcagatc cagccttgat attatgcat gaacttatac atgttttaca tggattatat 721 ggcattaag tagatgattt accaattgta ccaaatgaa aaaaattttt tatgcaatct 781 acagatgcta cagaggcaga agaactatat acatttggag cacaagatcc cagcatcata 841 actccttcta cggataaaag tatctatgat aaagttttgc aaaattttag agggatagtt 901 gatagactta acaaggtttt agtttgcata tcagatccta acattaatat fathers 961 aaaaataaat ttaagataa atataaattc gttgaagatt ctgagggaaa attatagtata 1021 gatgtagaaa gttttgataa attataataa agcttaaatgt ttggttttacagaaactaat 1081 atagcagaaa attataaat aaaaactaga gcttcttatt ttagtgattccttaccacca 1141 gtaaaaata aaaatttatt agataatgaa atctatacta tagaggaagggtttaatata 1201 tctgataaag atatggaaaa agaatataaga ggtcagaata aagctataaataacaagct 1261 gcatttggct gcaatgtgtaaaagtgtt 1321 aaagctccag gatatgtat tgatgttgat atgagatt tgttctttatagctgataaa 1381 atagtttt cagatgattt atctaaaaac gaagaatag atattacacacagagtaat 1441 tatatagaa atgactccc tattaatttag atactgatttaataagtaaa 1501 atagattac caagtgaaaa tacagaatca cttactgatt ttaatgtagatgttccagta 1561 tatgaaaaac aaccgctat aaaaaaatt tttacagatg aaataccatctttcaat 1621 ttatactc agacattcc tctagatata agagatata gtttaaccttcattgat 1681 gatgcattat tattttctaa caagtttat tcatttttt ctatggattattattaaaact 1741 gctaataag tggtagaagc aggattttt gcaggttggg tgaacagatagtaatgat 1801 ttgtaatcg aagctataa agcaatact atggataaa ttgcagatatatctctatt 1861 gttccttata taggattagc tttaatgta ggaatgaaa cagctaaggattttgaa 1921 aatgctttg agatgcagg agccagtatt ctactagaat ttataccagaacttttata 1981 cctgtagttg gagccttttt attagaatca tattattgaca aaaaaaaattattaaa 2041 acatagata atgctttaac taaaagaat gaaaatgga gtgatatgtacggattaata 2101 gtagcgcaat ggctctcac agttaatact caatttatta cataaaagagggaatgtat 2161 aaggcttta atttacaagc acaagcattg gagaata taaaatacagattatatata 2221 tattctgaa aaagaaagtc aattattac atcgatttta atgatataattctaactt 2281 atgaggta ttaccaagc tatagataat attaatt tattaatggatgttctgta 2341 tcatattaa tgaaaaaaat gattccatta gctgtagaaa attackagactttgaataat 2401 gctctcaaa aaaatttgtt aaattatata gatgaaaata attatatttgattggagt 2461 gcagaatatg aaaatcaa agtaaataa tacttgaaa ccattatgccgttgatctt 2521 tcaatatta ccaatgaac atactaata gaatgtttta aataatatagcgaatt 2581 fatherfather fatherfather fatherfather fatherfatherfatherfatherfatherfatherfatherfatherfather. 2641 tatggggcaa aggtagaggt atatgatgga gtcgagctta atgataaaatcaatttaaa 2701 ttactagtt cagcaatag windows gtgactcaa atcagaatcatttaat 2761 agtgtgttcc ttgatttag cgttagcttt tggatagaa tacctaaataataagaatgat 2821 gtatacaa attatattca tatgaat acaatatta attgtatgaaaaatattcg 2881 ggctggaaaa tatctattag gggtaatagg attatatgga ctttaattgatataatgga 2941 aaaccaaat cggtattttt tgaatagaac atagagaag atatatcagagtatattaat 3001 agatggtttt ttgtaactat tactaataat ttgaatacg ctaaaatttattaatggt 3061 aagctagaat cciatacaga tattaaagaat ttattgctaatggtgaata 3121 atatttaat tagaggtga tatagataga acacaattta ttggatgaataatttcagt 3181 atttttata cggattaag tcaatcaat attgagaa gattaaattcaatcatat 3241 agcgaatatt taaagattt ttggggaat cctttaatgt acaataagaatattattatg 3301 tttaatgcgg ggataaaaa ttcatatatt aaactaaga aagattcacctgtaggtgaa 3361 atttttacac gtagcaata taatcaaat tctaata taattatagagatttatat 3421 attggagaa atttattat aagaaag tcaatttctc attctataatgatgatata 3481 gttagaaaag aagattatat atatctagat ttttttaatt taaatcaagagtggagagta 3541 tatacctata aatattttaa gaaagaggaa gaaaaattgt ttttagctcctataagtgat 3601 tctgatgagt tttacaatac tatacaaata aaagaatatg atgaacagccaacatatagt 3661 tgtcagttgc tttttaaaaa agatgaagaa agtactgatg agataggattgattggtatt 3721 catcgtttct acgaatctgg aattgtattt gaagagtata aagattatttttgtataagt 3781 aaatggtact taaaagaggt aaaaaggaaa ccatataatt taaaattggggatgtaattgg 3841 cagtttattc ctaaagatga agggtggact gaataa c. LC / B coordinates, derived from protein structure PDB:1S0F, with added Met. N-end 1-17 MLD-R1 281-305 MLD-R2 306-340 MLD-R1-R2 280-340 LHD-R1-R2-R3 280-365 LC / B 1-437 BoNT / B 1291 3. BoNT / C a. Amino acid sequence LC / C 1-437 (SEQ ID NO: 19) 1 MPITINNFNY SDPVDNKNIL YLDTHLNTLA NEPEKAFRIT GNIWVIPDRF SRNSNPNLNK 61 PPRVTSPKSG YYDPNYLSTD SDKDTFLKEI IKLFKRINSR EIGEELIYRL STDIPFPGNN 121 NTPINTFDFD VDFNSVDVKT RQGNNWVKTG SINPSVIITG PRENIIDPET STFKLTNNTF 181 AAQEGFGALS IISISPRFML TYSNATNDVG EGRFSKSEFC MDPILILMHE LNHAMHNLYG 241 IAIPNDQTIS SVTSNIFYSQ YNVKLEYAEI YAFGGPTIDL IPKSARKYFE EKALDYYRSI 301 AKRLNSITTA NPSSFNKYIG EYKQKLIRKY RFVVESSGEV TVNRNKFVEL YNELTQIFTE 361 FNYAKIYNVQ NRKIYLSNVY TPVTANILDD NVYDIQNGFN IPKSNLNVLF MGQNLSRNPA 421 LRKVNPENML YLFTKFC a. Amino acid sequence BoNT / C1 1 - 1291 (SEQ ID NO: 20) 1 MPITINNFNY SDPVDNKNIL YLDTHLNTLA NEPEKAFRIT GNIWVIPDRF SRNSNPNLNK 61 PPRVTSPKSG YYDPNYLSTD SDKDTFLKEI IKLFKRINSR EIGEELIYRL STDIPFPGNN 121 NTPINTFDFD VDFNSVDVKT RQGNNWVKTG SINPSVIITG PRENIIDPET STFKLTNNTF 181 AAQEGFGALS IISISPRFML TYSNATNDVG EGRFSKSEFC MDPILILMHE LNHAMHNLYG 241 IAIPNDQTIS SVTSNIFYSQ YNVKLEYAEI YAFGGPTIDL IPKSARKYFE EKALDYYRSI 301 AKRLNSITTA NPSSFNKYIG EYKQKLIRKY RFVVESSGEV TVNRNKFVEL YNELTQIFTE 361 FNYAKIYNVQ NRKIYLSNVY TPVTANILDD NVYDIQNGFN IPKSNLNVLF MGQNLSRNPA 421 LRKVNPENML YLFTKFCHKA IDGRSLYNKT LDCRELLVKN TDLPFIGDIS DVKTDIFLRK 481 DINEETEVY YPDNVSVDQV ILSKNTSEHG QLDLLYPSID SESEILPGEN QVFYDNRTQN 541 VDYLNSYYYL ESQKLSDNVE DFTFTRSIEE ALDNSAKVYT YFPTLANKVN AGVQGGLFLM 601 WANDVVEDFT TNILRKDTLD KISDVSAIIP YIGPALNISN SVRRGNFTEA FAVTGVTILL 661 EAFPEFTIPA LGAFVIYSKV QERNEIIKTI DNCLEQRIKR WKDSYEWMMG TWLSRIITQF 721 NNISYQMYDS LNYQAGAIKA KIDLEYKKYS GSDKENIKSQ VENLKNSLDV KISEAMNNIN 781 KFIRECSVTY LFKNMLPKVI DELNEFDRNT KAKLINLIDS HNIILVGEVD KLKAKVNNSF 841 QNTIPFNIFS YTNNSLLKDI INEYFNNIND SKILSLQNRK NTLVDTSGYN AEVSEEGDVQ 901 LNPIFFPDFK LGSSGEDRGK VIVTQNENIV YNSMYESFSI SFWIRINKWV SNLPGYTIID 961 SVKNNSGWSI GIISNFLVFT LKQNEDSEQS INFSYDISNN APGYNKWFFV TVTNNMMGNM 1021 KIYINGKLID TIKVKELTGI NFSKTITFEI NKIPDTGLIT SDSDNINMWIRDFYIFAKEL 1081 DGKDINILFN SLQYTNVVKD YWGNDLRYNK EYYMVNIDYL NRYMYANSRQIVFNTRRNNN 1141DFNEGYKIII KRIRGNTNDT RVRGGDILYF DMTINNKAYN LFMKNETMYA DNHSTEDIYA 1201 IGLREQTKDI NDNIIFQIQP MNNTYYYASQ IFKSNFNGEN ISGICSIGTYRFRLGGDWYR 1261HNYLVPTVKQ GNYASLLEST STHWGFVPVS E b. DNA sequence BoNT / C (SEQ ID NO: 21) 1 atgccaataa caattaacaa ctttaattat tcagatcctg ttgataataa aaatatttta 61 tatttagata ctcatttaaa tacactagct aatgagcctg aaaaagcctt tcgcattaca 121 ggaaatatat gggtaatacc tgatagattt tcaagaaatt ctaatccaaa tttaaataaa 181 cctcctcgag ttacaagccc taaaagtggt tattatgatc ctaattattt gagtactgat 241 tctgacaaag atacattttt aaaagaaatt ataaagttat ttaaaagaat taattctaga 301 gaaataggag aagaattaat atatagatactt tcgacagata taccctttcc tgggaataac 361 atactccaa ttaatacttt tgattttgat gtagatttta acagtgttga tgttaaaact 421 agacaaggta acaactgggt taaaactggt agcataaatc ctagtgtta ataactgga 481 cctagagaaa acattataga tccagaact tctacgttta aattaacta caatactttt 541 gcggcacaag aaggattgg tgctttatca ataatttcaa tatcacctag atttatgcta 601 acatagta atgcaactaa tgatgtagga gagggtagat ttctaagtc tgaatttgc 661 atggatccaa tactatttt aatgcatgaa cttaatcatg caatgcataa tttatatgga 721 atagctatac caaatgatca aacaatttca tctgtaacta gtaatatttt ttattctca 781 tataatgtga aattagagta tgcagaaata tatgcattg gaggtccaac tatagacctt 841 attcctaaaa gtgcaggaa atattttgag gaaaaggcat tggatta tagatctata 901 gctaaagac ttaatagtat aactactgca aatccttca gctttaataa atatataggg 961 gatataac agaacttat tagagtat agatcgtag tagatctc aggtgaagtt 1021 acagtaatc gtaatagtt tgttgagtta tattaatgac ttacacaatttacagaa 1081 tttaactacg ctaaaata taatgtacaa ataggaaa tatatctttcaatgtatat 1141 actccggtta cggcgaat attagacgat aatgtttag atatacaaatggatttaat 1201 attackaaa gtaatttaaa tgtactattt atgggtcaa atttatctcgaatccagca 1261 ttagaaag tcaatcctga aaatatgctt tattttatta caaatttgtcataaagca 1321 atagatggta gatcattatta taaaaaca ttagattgta gagagctttagttaaaaat 1381 actgacttac cctttatagg tgatattagt gatgttaaaa ctgatatatttttaagaaaa 1441 gatttaatg aagaactga agttatatac tatccggaca atgtttcagtagatcaagtt 1501 attctcagta agaataccctc agaacatgga caacagatt tattacctagtattgac 1561 agtgagagtg aattattacc agggagaat caagtctttt atgatagaactcaaat 1621 gttgattatt tgaattctta attaccta gatctcaa aactaagtgataatgttgaa 1681 gattttactt tacgagatc attgaggag gctttggata atagtgcaaagtatact 1741 tactttccta cactagctaa taaagtaaat gcgggtgttc aaggtggttttttaatg 1801 tgggcaatg atgtagttga agatttact acaatattc windowagaatacattagat 1861 aaatatcag atgtatcagc tattattccc tatataggac ccgcattaaataatagtaat 1921 tctgtagaa gaggaattt tactgaagca tttgcagtta ctggtgtaactattatta 1981 gaagcattc ctgaatttac atacctgca cttggtgcat ttgtgatttatagtaggtt 2041 shaaaaaaaaaaaaaaaactared windows eagletgtt windowaaaaaaaah 2101 tggaagatt catatgaatg gatgatggga acgtggttat ccaggattattactcaattt 2161 aatata gttatcaat gtatgattct ttaattatc aggcaggtgcaatcaaagct 2221 aaatagatt tgaata aaaatattca ggagtgata aaaaaaaaagtca 2281 gttgaaaatt taaaaaatag tttagataaaaatttcgg aagcaatgaaaattaaat 2341 aattatac gagaatgttc cgtaacatat ttttaaaa atatgttacctaaagtatt 2401 gatgatta atgagtttga tcgaatact aaagcaaat tattaatcttatagattagt 2461 cataatatta ttctagttgg tgagtagat aaatttaaag aaagtaatatagcttt 2521 CAAATACA taccctttaa tattttttca tatactaata attctttaaaagatata 2581 attaatgat atttcataa attaatgat tcaaaattt tgagcctacaaaacagaaaaa 2641 atactttag tggatacatc aggatatat gcagaagtga gtgaagaggcgatgttcag 2701 cttaatccaa tattccatt tgactttaaa ttaggtagtt caggggatagaggtaaa 2761 gttatagtaa cccagaatga aaatattgta tataattcta tgtatgaagttttagcatt 2821 agttttgga ttagaataa taatgggta agtaatttac ctggatatactatattgat 2881 agtgttaaaa atactcagg ttggagtata gtattatta gtattttttttacttact 2941 ttaaaaaaaaatgaatag tgaaaaaaatttta gttagattaatcaataat 3001 gctcctggat acaataaatg gttttttgta actgttacta acaatgatgatgggaatg 3061 aagattatta taaatggaa attaatagat actataaag ttaagaactactggatt 3121 attttagca aaactataac atttgaata aaaaattc cagataccggtttgattact 3181 tcagattctg atacatcaa tatgtggata agagatttt atatatttgctaagaatta 3241 gatggtaaag atattaatat attatttaat agcttgcaat atactaatgttgtaaaagat 3301 tattggggaa atgatttaag atataataaa gaatattata tggttaatatagattattta 3361 aatagatata tgtatgcgaa ctcacgacaa attgttttta atacacgtagaaataataat 3421 gacttcaatg aaggatataa aattataata aaaagaatca gaggaaatacaaatgatact 3481 agagtacgag gaggagatat tttatatttt gatatgacaa ttaataacaaagcatataat 3541 ttgtttatga agaatgaaac tatgtatgca gataatcata gtactgaagatatatatgct 3601 ataggtttaa gagaacaaac aaaggatata aatgataata ttatatttcaaatacaacca 3661 atgaataata cttattatta cgcatctcaa atatttaaat caaattttaatggagaaaat 3721 atttctggaa tatgttcaat aggtacttat cgttttagac ttggaggtgattggtataga 3781 cacaattatt tggtgcctac tgtgaagcaa ggaaattatg cttcattattagaatcaaca - c.LC / C: Derived from the crystal structure c.LC / C:来源于晶体结构 PDB:2QN0 Coordinates N - terminus 1 - 17 MLD - R1 284 - 305 MLD-R2 306-342 MLD-R1-R2 283-343 LHD-R1-R2-R3 283-367 LC / C1 1-437 BoNT / C1 1291 4. BoNT / D a. Amino acid sequence LC / D 1-437 (SEQ ID NO: 22) 1 MTWPVKDFNY SDPVNDNDIL YLRIPQNKLI TTPVKAFMIT QNIWVIPERF SSDTNPSLSK 61 PPRPTSKYQS YYDPSYLSTD EQKDTFLKGI IKLFKRINER DIGKKLINYL VVGSPFMGDS 121 STPEDTFDFT RHTTNIAVEK FENGSWKVTN IITPSVLIFG PLPNILDYTA SLTLQGQQSN 181 PSFEGFGTLS ILKVAPEFLL TFSDVTSNQS SAVLGKSIFC MDPVIALMHE LTHSLHQLYG 241 INIPSDKRIR PQVSEGFFSQ DGPNVQFEEL YTFGGLDVEI IPQIERSQLR EKALGHYKDI 301 AKRLNNINKT IPSSWISNID KYKKIFSEKY NFDKDNTGNF VVNIDKFNSL YSDLTNVMSE 361 VVYSSQYNVK NRTHYFSRHY LPVFANILDD NIYTIRDGFN LTNKGFNIEN SGQNIERNPA 421 LQKLSSESVV DLFTKVC a. Amino acid sequence BoNT / D 1-1276 (SEQ ID NO: 23) 1 MTWPVKDFNY SDPVNDNDIL YLRIPQNKLI TTPVKAFMIT QNIWVIPERF SSDTNPSLSK 61 PPRPTSKYQS YYDPSYLSTD EQKDTFLKGI IKLFKRINER DIGKKLINYL VVGSPFMGDS 121 STPEDTFDFT RHTTNIAVEK FENGSWKVTN IITPSVLIFG PLPNILDYTA SLTLQGQQSN 181 PSFEGFGTLS ILKVAPEFLL TFSDVTSNQS SAVLGKSIFC MDPVIALMHE LTHSLHQLYG 241 INIPSDKRIR PQVSEGFFSQ DGPNVQFEEL YTFGGLDVEI IPQIERSQLR EKALGHYKDI 301 AKRLNNINKT IPSSWISNID KYKKIFSEKY NFDKDNTGNF VVNIDKFNSL YSDLTNVMSE 361 VVYSSQYNVK NRTHYFSRHY LPVFANILDD NIYTIRDGFN LTNKGFNIEN SGQNIERNPA 421 LQKLSSESVV DLFTKVCLRL TKNSRDDSTC IKVKNNRLPY VADKDSISQE IFENKIITDE 481 TNVQNYSDKF SLDESILDGQ VPINPEIVDP LLPNVNMEPL NLPGEEIVFY DDITKYVDYL 541 NSYYYLESQK LSNNVENITL TTSVEEALGY SNKIYTFLPS LAEKVNKGVQ AGLFLNWANE 601 VVEDFTTNIM KKDTLDKISD VSVIIPYIGP ALNIGNSALR GNFNQAFATA GVAFLLEGFP 661 EFTIPALGVF TFYSSIQERE KIIKTIENCL EQRVKRWKDS YQWMVSNWLS RITTQFNHIN 721 YQMYDSLSYQ ADAIKAKIDL EYKKYSGSDK ENIKSQVENL KNSLDVKISE AMNNINKFIR 781 ECSVTYLFKN MLPKVIDELN KFDLRTKTEL INLIDSHNII LVGEVDRLKA KVNESFENTM 841 PFNIFSYTNN SLLKDIINEY FNSINDSKIL SLQNKKNALV DTSGYNAEVR VGDNVQLNTI 901 YTNDFKLSSS GDKIIVNLNN NILYSAIYEN SSVSFWIKIS KDLTNSHNEY TIINSIEQNS 961 GWKLCIRNGN IEWILQDVNR KYKSLIFDYS ESLSHTGYTN KWFFVTITNN IMGYMKLYIN 1021 GELKQSQKIE DLDEVKLDKT IVFGIDENID ENQMLWIRDF NIFSKELSNEDINIVYEGQI 1081 LRNVIKDYWG NPLKFDTEYY IINDNYIDRY IAPESNVLVL VQYPDRSKLYTGNPITIKSV 1141 SDKNPYSRIL NGDNIILHML YNSRKYMIIR DTDTIYATQG GECSQNCVYALKLQSNLGNY 1201 GIGIFSIKNI VSKNKYCSQI FSSFRENTML LADIYKPWRF SFKNAYTPVAVTNYETKLLS 1261 TSSFWKFISR DPGWVE b. DNA sequence BoNT / D (SEQ ID NO: 24) 1 atgacatggc cagtaaaaga ttttaattat agtgatcctg ttaatgacaa tgatatatta 61 tatttaagaa taccacaaaa taagttaatt actacacctg taaaagcttt tatgattact 121 caaaatattt gggtaatacc agaaagattt tcatcagata ctaatccaag tttaagtaaa 181 ccgcccagac ctactcaaa gtatcaaagt tattatgatc ctagttattt atctactgat 241 gaaaaaag attackttt aaagggatt aaaaattt t'aaaagaat t'aaaaga 301 gatataggaaaaaattaat aaatttatta gtagttggtt caccttttat gggagattca 361 agtacgcctg agatacatt tgattttaca cgtcatacta ctaatattgc agttgaaag 421 ttgaaatg gtagttggaa agtaacaat attatacac caagtgtatt gatatttgga 481 ccactccta atatattaga ctatacagca tccttacat tgcaggaca acaatcaat 541 ccatcattg aagggttgg aacattatct attackaaag tagcacctga atttttgtta 601 acatttagtg atgtaacatc taatcaagt tcagctgtat taggcaatc tatattttgt 661 atggatccag taatagcttt atgcatgag ttaacacatt ctttgcatca attatatgga 721 aataatac catctgataa aaggattcgt ccacagtta gcgaggtt tttctca 781 gatgaccca acgtacaatt tgaggaatta tatacattg gaggattaga tgttgaata 841 atacctcaaa tgaagatc aattaag gaaaaagcat taggtcacta in windows 901 gcgaaagac tatatatat taataaact attccttcta gttggattag tatatagat 961 aaataaa aaatattttc tgaaagtat aatttgata agaataac aggaattttt 1021 gttgtaaata ttgataaatt caatagctta tattcagact tgactaatgttatgtcagaa 1081 gttgttatt cttcgcaata taatgttaa aacaggactc attatttttcaggcattat 1141 ctacctgtat ttgcaatat attagatgat atatttata ctataagagatggttttaat 1201 ttaaaata aaggttttaa tatagaaaat tcggtcaga atagaaggaatcctgca 1261 ctacaaagc ttagttcaga aagtgtagta gatttatta caaagtgtttagatta 1321 aaaaaata gtagagatga ttcaacatgt atttaagtta aaaatagattaccttat 1381 gtagctgata agatagcat ttcacaagaa atatttgaa aaaattacagatgag 1441 actaatgtac aaaattattc agataaattt tcattagatg atctattttagatgggcaa 1501 gttcctatta atcctgaaat agtagatcca ctattaccca atgttaatatggaaccttta 1561 atcttccag gtgaagaat agtattttat gatgatatta ctaaatatgttgattta 1621 attcttatt attatttgga atctcaaaa ttaagtaata atgttgaaatattactctt 1681 acacttcag ttgagaagc attaggttat agcaataga tatacacattttttacctagc 1741 tagctgaa aagtgaataa agtgttcaa gcaggtttat tcttaattgggcgaatgaa 1801 gtagttgagg atttactac aattatg aagaagata cattgataaatcagat 1861 gtatcagta taattccatta taggact gccttaata taggaattcagcattaagg 1921 ggaaattta atcaagcatt tgcacagct ggtgtagctt ttttagagggatttcca 1981 gagtttacta tacctgcact cggtgtattt accttta gttctattcaagaagagag 2041 aaaattatta aaactataga aattgtttg gaaaagag ttaagagaagattca 2101 tatcaatgga tgtatcaa tggttgtca agattacta ctcaatttaatcatataaat 2161 tatcaatgt atgatcttt aagttatcag gcagatgcaa tcaagctaaatagattta 2221 gatataaa atactcagg agtgaataa gatataaaagtcaagttgaaattta 2281 aaaatagtt tagagtaa aatttcggaa gcaatgaata ataataatttatacga 2341 gaatgttctg taacatactt atttaaaaat atgctcccta aagtaattgacgaattaaat 2401 aagtttgatt taagaactaa aacagaatta attaatctta tagagatcataatattatt 2461 ctagttggtg aagtatag attaaaagca aaagtaaatg agagttttgaaaatacaatg 2521 ccttttaata ttttttcata tactaataat tctttattaa aagatataattaatgaatat 2581 ttcaatagta ttaatgattc aaaaattttg agcttacaaa acaaaaaaaatgctttagtg 2641 gatacatcag gatataatgc agaagtgagg gtaggagata atgttcaacttaatacgata 2701 tatacaaatg actttaaatt aagtagttca ggagataaa ttatagtaaatttaaat 2761 aatattttat atagcgctat ttatgagaac tctagtgtta gtttttggattaagatatct 2821 aaagatttaa ctaattctca taatgaatat acaataatta acagtatagaacaaaattct 2881 gggtggaaat tatgtattag gaatggcaat atagaatgga tttatacaagatgttaataga 2941 aagtataaaaa gtttaatttt tgattatagt gaatcattaa gtcatacaggatatacaaat 3001 aaatggtttt ttgttactat aactaataat ataatggggt atatgaaactttatataaat 3061 Gaggattaa agcagagtca aaaaattgaa gatttagatg agggttaagttaaaacc 3121 atagtatttg gatagatga gatatagat gagaatcaga tgctttggattagatttt 3181 Attattttt ctaagaatt agtaatgaa gatttatta ttgtatatgagggacaata 3241 ttaagaatg ttattaaga ttattgggga atccttttga agtttgatacagaatattat 3301 attattaatg attattatat agatagtat atagcacctg aaagtaatgtacttgtactt 3361 gttcagtatc cagatagatc taaattatat actggaatc ctattactattaatcagta 3421 tctgataga atccttatag taggatttta atggata atatattctcatatgtta 3481 crowded gaatattatagatagata chattattatgshaagga 3541 ggagtgtt cacaaattg tgtatatgca ttaaaattac agagtaatttaggtattat 3601 ggtataggta tattagtat aaaaaatatt gtactaaaaaatattgtagtcaatt 3661 ttcttagtt ttagggaaa tacaatgctt ctagcagata tataaaccttggattt 3721 tctttaaaa atgcatacac gccagttgca gtaactatt atgaacaaactattaca 3781 acttcatctt tttggaaatt tatttctagg gatccaggat gggtagagta a c.LC / D coordinates, derived from the crystal structure PDB:2FPQ Chapter 1-18 MLD-R1 284-307 MLD-R2 308-342 MLD-R1-R2 284-342 LHD-R1-R2-R3 284-367 LC / D 1-437 BoNT / D 1276 5. BoNT / F a. Amino acid sequence LC / F 1-429 (SEQ ID NO: 25) 1 MPVVINSFNY NDPVNDDTIL YMQIPYEEKS KKYYKAFEIM RNVWIIPERN TIGTDPSDFD 61 PPASLENGSS AYYDPNYLTT DAEKDRYLKT TIKLFKRINS NPAGEVLLQE ISYAKPYLGN 121 EHTPINEFHP VTRTTSVNIK SSTNVKSSII LNLLVLGAGP DIFENSSYPV RKLMDSGGVY 181 DPSNDGGFSI NIVTFSPEYE YTFNDISGGY NSSTESFIAD PAISLAHELI HALHGLYGAR 241 GVTYKETIKV KQAPLMIAEK PIRLEEFLTF GGQDLNIITS AMKEKIYNNL LANYEKIATR 301 FNEIYKKLYS FTEIDLANKF LSRVNSAPPE IDINEYKDYF QKYGLDKNA 361 KVKCRNTYFI KYGFLKVPNL LDDDIYTVSE GFNIGNLAVN NRGQNIKLNP KIIDSIPDK 421 LVEKIVKFC a. Amino acid sequence BoNT / F 1-1278 (SEQ ID NO: 26) 1 MPVVINSFNY NDPVNDDTIL YMQIPYEEKS KKYYKAFEIM RNVWIIPERN TIGTDPSDFD 61 PPASLENGSS AYYDPNYLTT DAEKDRYLKT TIKLFKRINS NPAGEVLLQE ISYAKPYLGN 121 EHTPINEFHP VTRTTSVNIK SSTNVKSSII LNLLVLGAGP DIFENSSYPV RKLMDSGGVY[[ID=II]] 181 DPSNDGFGSI NIVTFSPEYE YTFNDISGGY NSSTESFIAD PAISLAHELI HALHGLYGAR 241 GVTYKETIKV KQAPLMIAEK PIRLEEFLTF GGQDLNIITS AMKEKIYNNL LANYEKIATR 301 LSRVNSAPPE YDINEYKDYF QWKYGLDKNA DGSYTVNENK FNEIYKKLYS FTEIDLANKF 361 KVKCRNTYFI KYGFLKVPNL LDDDIYTVSE GFNIGNLAVN NRGQNIKLNP KIIDSIPDKG 421 LVEKIVKFCK SVIPRKGTKA PPRLCIRVNN RELFFVASES SYNENDINTP KEIDDTTNLN 481 NNYRNNLDEV ILDYNSETIP QISNQTLNTL VQDDSYVPRY DSNGTSEIEE HNVVDLNVFF[[ID=I]] 541 YLHAQKVPEG ETNISLTSSI DTALSEESQV YTFFSSEFIN TINKPVHAAL FISWINQVIR 601 DFTTEATQKS TFDKIADISL VVPYVGLALN IGNEVQKENF KEAFELLGAG ILLEFVPELL 661 IPTILVFTIK SFIGSSENKN KIIKAINNSL MERETKWKEI YSWIVSNWLT RINTQFNKRK 721 EQMYQALQNQ VDAIKTVIEY KYNNYTSDER NRLESEYNIN NIREELNKKV SLAMENIERF 781 ITESSIFYLM KLINEAKVSK LREYDEGVKE YLLDYISEHR SILGNSVQEL NDLVTSTLNN 841 SIPFELSSYT NDKILILYFN KLYKKIKDNS ILDMRYENNK FIDISGYGSN ISINGDVYIY 901 STNRNQFGIY SSKPSEVNIA QNNDIIYNGR YQNFSISFWV RIPKYFNKVN LNNEYTIIDC 961 IRNNNSGWKI SLNYNKIIWT LQDTAGNNQK LVFNYTQMIS ISDYINKWIF VTITNNRLGN 1021 SRIYINGNLI DEKSISNLGD IHVSDNILFK IVGCNDTRYV GIRYFKVFDTELGKTEIETL 1081 YSDEPDPSIL KDFWGNYLLY NKRYYLLNLL RTDKSITQNS NFLNINQQRGVYQKPNIFSN 1141 TRLYTGVEVI IRKNGSTDIS NTDNFVRKND LAYINVVDRD VEYRLYADISIAKPEKIIKL 1201 IRTSNSNNSL GQIIVMDSIG NNCTMNFQNN NGGNIGLLGF HSNNLVASSWYYNNIRKNTS 1261 SNGCFWSFIS KEHGWQEN b. DNA sequence BoNT / F (SEQ ID NO: 27) 1 atgccagttg taataatag ttttaatt atgaccctg ttaatgatga tacaattta 61 tacatgcaga taccatatga agaaaaaagt aaaaatatt aaagcttt tgagatttag 121 cgtaatgttt ggataattcc tgagagaat acataggaa cggatcctag tgatttgat 181 ccaccggctt cattagagaa cggaagcagt gcttatg atcctatta ttataccact 241 gatgctgaaa agatagata ttaaaaca acgataaaat tattaagag attaatagt 301 atatcctgcag gggaagtttt gttacaagaa atatcatatg ctaaaccata tttaggaat 361 gaacacacgc cattaatga attccatcca gttactagaa ctacaagtgt taatataaaa 421 tcatcaacta atgttaaag ttcaataata ttgaatcttc tgtattggg agcaggacct 481 gatatattg aaaattctc taccccgtt agaaactaa tggattcagg tggatttat 541 gacccaagta atgatggttt tggattcatt atatcgtga cattttcacc tgaatatgaa 601 tatacttta atgatattag tggaggtat aacagtagta cagaatcatt tattgcagat 661 cctgcaattt cactagctca tgaattgata catgcactgc atggatta cggggctagg 721 ggagttactt aagagac tataaaagta aagcaagcac ctcttatgat agccgaaaaa 781 cccataggc taggaatt tttaacctt ggaggtcagg atttaatat tattactt 841 gctatgagg aaaaaatata taacaatctt ttagctact atgaaaaaat agctactaga 901 cttagtagag ttaatagtgc tcctcctgaa tatgatatta atgaatataa agattatttt 961 caatggaagt atgggctaga taaaaatgct catggagatt atactgtaaa tgaaaaaaa 1021 tttaatgaaa tttataaaaa attatatagc tttacagaga ttgacttagcaaayattt 1081 aaagtaaat gtagaaatac ttattttatt aaatatggat ttttaaagttccaaatttg 1141 ttagatgatg atatttatac tgtatcagag gggtttaata taggtaatttagcagtaac 1201 aatcgcggac aaaatataa gttaaatcct aaattattg attccattccagataaggt 1261 ctagtggaa agatcgttaa atttgtaag agcgttattc ctagaaaaggtacaaggcg 1321 ccaccgcgac tatgcattag agtaaataat agggagttat tttgtagcttcagaagt 1381 agctataatg aaatgatat tatacacct aaagaaattg acgatacaacaatctaat 1441 attattatta gaataattt agatgaagtt attagatt attaagtgagacaataccct 1501 aaatca atcaacatt aaatacactt gtacaagacg atagttatgtgccagat 1561 gattctaatg gacaagtga atagaggaa cataatgttg ttgaccttaatgtatttttc 1621 tattacatg cacaaagt accagaggt gaactaata tagtttaacttcttcatt 1681 gatacggcat tatcagaga atcgcaagta tatacattct tttctcagagtttaat 1741 actacaata aacctgtaca cgcagcacta tttataagtt ggataaatcaagtaaga 1801 gattttacta ctgaagctac aaaaaagt actttgata agatgcagacatactcttta 1861 gttgtaccat atgtagtct tgctttaat atatagtaatg aggtacaaaagaaaatttt 1921 Aggaggcatt Aggaggggtt Attattag AatttGtGccagagcttta 1981 attcctaca ttttagtgtt tacataaaa tcctttatag gttcatctgagataaaaat 2041 aaatcatta aagcaataaaattcatta atggaagg aaaaaagtgaaagaata 2101 tagttgga tagtatcaa ttggctct agattata cacaatttaaaaaaaaa 2161 gaaaatgt atcaagcttt gcaaatca gtagatgca taaaaacagtaatagaat 2221 aadadatattatacttc agatgagaga atagacttg atctgaatadadcaat 2281 atatagag aagaattgaa aaaaagtt tctttagcaa tggaaatagagagattt 2341 atacagaga gttctatatt ttatttaatg agttaata atgaagccaagttagttaaa 2401 ttagagaat atgatgaagg cgttaaggaa tattgctag actatatttcagacataga 2461 tcaatttag gaatagtgt acagaatta atgatttag tgactagtactctgaat 2521 agtattccat ttgaactttc ttcatatact atgataaa ttctaattttatattttaat 2581 aattatata aaaaattta agatactct attattagata tgcgatatgaaaataaaa 2641 tttatagata tctctggata tggttcaat ataagcatta atggagatgtatatatttat 2701 tcaaata gaatcaatt tggaatatat agtagtagc ctagtgaagttaatagct 2761 caaataatg atattatata caatggtaga tatcaaatt ttagtagttttctgggta 2821 aggattccta atacttca taagtgaat cttaataatg atatactataatagatgt 2881 attaggaata attattcagg attattaattcactatttattattattattattaatttggact 2941 ttcagata ctgctggaaa taatcaaaa ctagtttta attatacacacaatgattagt 3001 atatctgatt atataata atgatttt gtaactatta ctaatatagattaggcaat 3061 tctagaattt acatcaatgg aaatttaata gatgaaaat caatttcgaatttaggtgat 3121 attcatgtta gtgataatat attatttaaa attgttggtt gtaatgatacaagatatgtt 3181 gtatagaat atttttaagt ttttgaacg gattagta aacagaattgagacttta 3241 tatagtgatg agccagatcc aagtatctta aaagacttt ggggaattattttgttatat 3301 aaaaagat atttattatt gatttacta agaacagata agtctattactcagaattca 3361 aactttctaa atattaatca aaagaggt gtttatcaga aaccaatattttttccac 3421 actagattat atacaggagt agagttatt atagaaaaa atggatctacagatatatct 3481 atacagata atttgttag aaaaatgat ctggcatata ttaatgtagtagatcgtgat 3541 gtagaatatc ggctatatgc tgatatatca attgcaaac cagagaaaataaaatta 3601 ataagaacat ctaattcaaa caatagctta ggtcaaatta tagttatggattcaatagga 3661 aatattgca caatgaattt tcaaaacaat aatgggggca aatataggattactaggtttt 3721 cats atttggttgc cattagttgg cats cats atttggttgc 3781 agtaatggat gcttttggag ttttatttct aaagagcatg gatggcaaga aaactaa c.LC / F cycle: Individual cycle cycle PDB:2A8A N-protein 1-1 MLD-R1 281–302 MLD-R2 303–336 MLD-R1-R2 280-336 LHD-R1-R2-R3 283-360 LC / F 1-429 BoNT / F 6. BoNT / G a. PHYSIOLOGY LC / G 1-436 (SEQ ID NO: 28) 1 MPVNIKXFNY NDPINNDDII MMEPFNDPGP GTYYKAFRII DRIWIVPERF TYGFQPDQFN 61 ASTGVFSKDV YEYYDPTYLK TDAEKDKFLK TMIKLFNRIN SKPSGQRLLD MIVDAIPYLG 121 NASTPPDKFA ADVANVSINK KIIQPGAEDQ IKGLMTNLII FGPGPVLSDN FTDSMIMNGH 181 SPISEGFGAR MMIRFCPSCL NVFNNVQENK DTSIFSRRAY FADPALTLMH ELIHVLHGLY 241 GIKISNLPIT PNTKEFFMQH SDPVQAEELY TFGGHDPSVI SPSTDMNIYN KALQNFQDIA 301 NRLNIVSSAQ GSGIDISLYK QIYKNKYDFV EDPNGKYSVD KDKFDKLYKA LMFGFTETNL 361 AGEYGIKTRY SYFSEYLPPI KTEKLLDNTI YTQNEGFNIA SKNLKTEFNG QNKAVNKEAY 421 EEISLEHLVI YRIAMC b. Amino acid sequence BoNT / G 1-1278 (SEQ ID NO: 29) 1 MPVNIKXFNY NDPINNDDII MMEPFNDPGP GTYYKAFRII DRIWIVPERF TYGFQPDQFN 61 ASTGVFSKDV YEYYDPTYLK TDAEKDKFLK TMIKLFNRIN SKPSGQRLLD MIVDAIPYLG 121 NASTPPDKFA ANVANVSINK KIIQPGAEDQ IKGLMTNLII FGPGPVLSDN FTDSMIMNGH 181 SPISEGFGAR MMIRFCPSCL NVFNNVQENK DTSIFSRRAY FADPALTLMH ELIHVLHGLY 241 GIKISNLPIT PNTKEFFMQH SDPVQAEELY TFGGHDPSVI SPSTDMNIYN KALQNFQDIA 301 NRLNIVSSAQ GSGIDISLYK QIYKNKYDFV EDPNGKYSVD KDKFDKLYKA LMFGFTETNL 361 AGEYGIKTRY SYFSEYLPPI KTEKLLDNTI YTQNEGFNIA SKNLKTEFNG QNKAVNKEAY 421 EEISLEHLVI YRIAMCKPVM YKNTGKSEQC IIVNNEDLFF IANKDSFSKD LAKAETIAYN 481 TQNNTIENNF SIDQLILDND LSSGIDLPNE NTEPFTNFDD IDIPVYIKQS ALKKIFVDGD 541 SLFEYLHAQT FPSNIENLQL TNSLNDALRN NNKVYTFFST NLVEKANTVV GASLFVNWVK 601 GVIDDFTSES TQKSTIDKVS DVSIIIPYIG PALNVGNETA KENFKNAFEI GGAAILMEFI 661 PELIVPIVGF FTLESYVGNK GHIIMTISNA LKKRDQKWTD MYGLIVSQWL STVNTQFYTI 721 KERMYNALNN QSQAIEKIIE DQYNRYSEED KMNINIDFND IDFKLNQSIN LAINNIDDFI 781 NQCSISYLMN RMIPLAVKKL KDFDDNLKRD LLEYIDTNEL YLLDEVNILK SKVNRHLKDS 841 IPFDLSLYTK DTILIQVFNN YISNISSNAI LSLSYRGGRL IDSSGYGATM NVGSDVIFND 901 IGNGQFKLNN SENSNITAHQ SKFVVYDSMF DNFSINFWVR TPKYNNNDIQ TYLQNEYTII 961 SCIKNDSGWK VSIKGNRIIW TLIDVNAKSK SIFFEYSIKD NISDYINKWF SITITNDRLG 1021 NANIYINGSL KKSEKILNLD RINSSNDIDF KLINCTDTTK FVWIKDFNIFGRELNATEVS 1081 SLYWIQSSTN TLKDFWGNPL RYDTQYYLFN QGMQNIYIKY FSKASMGETAPRTNFNNAAI 1141 NYQNLYLGLR FIIKKASNSR NINNDNIVRE GDYIYLNIDN ISDESYRVYVLVNSKEIQTQ 1201 LFLAPINDDP TFYDVLQIKK YYEKTTYNCQ ILCEKDTKTF GLFGIGKFVKDYGYVWDTYD 1261 NYFCISQWYL RRISENINKL RLGCNWQFIP VDEGWTE b. DNA sequence BoNT / G (SEQ ID NO: 30) 1 atgccagtta atataaaaan ctttaattat aatgacccta ttaataatga tgacattatt 61 atgatggaac cattcaatga cccagggcca ggaacatatt ataaagcttt taggattata 121 gatcgtattt ggatagtacc agaaaggttt acttatggat ttcaacctga ccaatttaat 181 gccagtacag gagtttttag taaagatgtc tacgaatatt acgatccaac ttatttaaaa 241 accgatgctg aaaaagataa atttttaaaa acaatgatta aattatttaa tagaattaat 301 tcaaaaccat caggacagag attactggat atgatagtag atgctatacc ttatcttgga 361 aatgcatcta caccgcccga caaatttgca gcaaatgttg caaatgtatc tattaataaa 421 aaaattatcc aacctggagc tgaagatcaa ataaaaggtt taatgacaaa tttaataata 481 tttggaccag gaccagttct aagtgataat tttactgata gtatgattat gaatggccat 541 tccccaatat cagaaggatt tggtgcaaga atgatgataa gattttgtcc tagttgttta 601 aatgtattta ataatgttca ggaaaataaa gatacatcta tatttagtag acgcgcgtat 661 tttgcagatc cagctctaac gttaatgcat gaacttatac atgtgttaca tggattatat 721 ggaattaaga taagtaattt accaattact ccaaatacaa aagaatttt catgcaacat 781 agcgatcctg tacaagcaga agaactatat acattcggag gacatgatcc tagtgttata 841 agtccttcta cggatatgaa tatttataat aaagcgttac aaaattttca agatatagct 901 aataggctta atattgtttc aagtgcccaa gggagtggaa ttgatatttc cttatataaa 961 caaatatata aaaataaata tgattttgtt gaagatccta atggaaaata tagtgtagat 1021 aaggataagt ttgataaatt atataaggcc ttaatgtttg gctttactgaaactaatcta 1081 gctggtgaat atggaataaa aactaggtat tcttatttta gtgaatatttgccaccgata 1141 aaaactgaaa aattgttaga caatacaatt tatactcaaa atgaaggctttaacatagct 1201 agtaaaaatc tcaaaacgga atttaatggt cagaataagg cggtaaataaaagaggcttat 1261 gagaaatca gcctagaaca tctcgttata tatagatag caatgtgcaagcctgtaatg 1321 tacaaaaata ccggtaaatc tgaacagtgt attattgtta atatgaggattttttttc 1381 atagctaata aagatagtttt ttcaaagat ttagctaag cagaactatagcatataat 1441 acaaaata atactataga aaatatttt tctatagatc agttgattttagataatgat 1501 ttagcagtg gcatagactt accaaatgaaacacagaac catttacaatttttgacgac 1561 atagatatcc ctgtgtatat taaacaatct gctttaaaaa aaattttgtggatggagat 1621 agccttttg atattaca tgctcaaca ttccttcta atagaaaatctacaacta 1681 acgaattcat taaatgatgc tttaagaat ataataag tctatactttttttctaca 1741 aaccttgttg aaaagctaa tacagttgta gtgctcac ttttgtaactgggtaaaa 1801 GTATATAG ATGATTTAC ATCTGAATCC ACCAAAAAAA GTACTTAGATAAGTTCA 1861 gatgtatcca taattattcc ctatatagga cctgctttga atgtaggaatgaacagct 1921 aaagaaatt ttaaaaatgc ttttgaata ggtggagccg ctatcttaatggattttatt 1981 Tccagaactta Tgtacctat Agttgatt Tttacattag AtcatgTaggaaaaaa 2041 gggcatatta tttgacgat atccaatgct ttaagaaaa gggatcaaaatggacagat 2101 atgtatggtt tgatagtatc gcagtggctc tcaacggtta atactcaattttatacaata 2161 aaaagaa tgtacaatgc ttataataat catcacaag caatagaaaaaatagaa 2221 gatataata atagatatag tgagagat aaatgaata ttācattgatttaatgat 2281 atagatttta aacttaatca agtataat tagcaata acaatagatgattta 2341 aaccaatgtt ctatatcata tctaatgaat agaatgatc cattagctgtaaaaagtta 2401 aaagactttg atgataatct tagagagat ttattggagt attagatacaatgaacta 2461 tattactg atgagtaa tattctaaa tcaaagtaa atagacacctaaagacagt 2521 ataccattg atctttcact ataccaccag gacaattt tatacaagttttttaat 2581 tattagtta attagtag taatgctatt ttaagtttta gttatagaggtggcgtttta 2641 atagattcat ctggatatgg tgcaactatg aatgtaggtt cagatgttatctttaatgat 2701 ataggaatg gtcaatttaa attaataat tctgaaata gtatattacggcacatcaa 2761 agtaaattcg ttgtatatga tagtatgtttt gatattttta gcattaacttttgggtagg 2821 actcctaaat attaata tgatatacaa acttatctc aaaatgagtatacaatatt 2881 agttgtataa aaatgactc aggatggaa gtatctatta agggaatagaatatatgg 2941 acattaatag atgttaatgc aaaatctaaa tcaatatttt tcgaatagtataaaagat 3001 attatatcag attattaaa taaatggttt tcataacta ttactaatgatagattaggt 3061 aacgcaata tttataaa tggagttg aaaaaagtg aaaaaatttaacttagat 3121 agaatttatt ctagtaatga tatagacttc aattatta attgtacagatactaaa 3181 ttgtttgga ttaaggattt taatatttt ggtagagaat taatgctacagaagtatctct 3241 tcactatatt ggattcaatc atctacaaat actttaaag atttttgggggaatcctttta 3301 agatacgata cacaatacta tctgtttaat caagtagc aaatatctatataagtat 3361 tttagtaag cttctatggg ggaactgca ccacgtacaa actttaataatgcagcaata 3421 aattatcaaa atttatatct tggtttacga tttattataa aaaaagcatcaaattctcgg 3481 aatataaata atgataatat agtcagagaa ggagattata tatatcttaatattgataat 3541 atttctgatg aatcttacag agtatatgtt ttggtgaatt ctaaagaaattcaaactcaa 3601 ttatttttag cacccataaa tgatgatcct acgttctatg atgtactacaaataaaaaaa 3661 tattatgaaa aaacaacata taattgtcag atactttgcg aaaaagatactaaaacattt 3721 gggctgtttg gaattggtaa atttgttaaa gattatggat atgtttgggatacctatgat 3781 aattattttt gcataagtca gtggtatctc agaagaatat ctgaaaatataaataaatta 3841 aggttgggat gtaattggca attcattccc gtggatgaag gatggacaga ataa c. LC / G coordinates, derived from the crystal structure PDB:1ZB7 <​​​​​​​​​​​​​​​​​​​​​​

[0396] 7. BoNT / A3

[0397] a. Amino acid sequence LC / A3 1-426 (SEQ ID NO: 31)

[0398] 1 MPFVNKQFNY RDPVNGVDIA YIKIPNAGQM QPVKAFKIHE GVWVIPERDT FTNPEEGDLN

[0399] 61 PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VIKLFDRIYS TGLGRMLLSF IVKGIPFWGG

[0400] 121 STIDTELKVI DTNCINVIEP GGSYRSEELN LVITGPSADI IQFECKSFGH DVFNLTRNGY

[0401] 181 GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GTFATDPAVT LAHELIHAAH RLYGIAINPN

[0402] 241 RVLKVKTNAY YEMSGLEVSF EELRTFGGND TNFIDSLWQK KFSRDAYDNL QNIARILNEA

[0403] 301 KTIVGTTTPL QYMKNIFIRK YFLSDASGK ISVNKPAFKE FYRVLTRGFT ELEFVNPFKV

[0404] 361 INRKTYNLNFD KAVFRINIVP DENYTINEGF NLEGANSNGQ NTEINSRNFT RLKNFTGLF

[0405] 421 FYKLLC

[0406] b. Amino acid sequence BoNT / A3 1-1292 (SEQ ID NO: 32)

[0407] 1 MPFVNKQFNY RDPVNGVDIA YIKIPNAGQM QPVKAFKIHE GVWVIPERDT FTNPEEGDLN

[0408] 61 PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VIKLFDRIYS TGLGRMLLSF IVKGIPFWGG

[0409] 121 STIDTELKVI DTNCINVIEP GGSYRSEELN LVITGPSADI IQFECKSFGH DVFNLTRNGY

[0410] 181 GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GTFATDPAVT LAHELIHAAH RLYGIAINPN

[0411] 241 RVLKVKTNAY YEMSGLEVSF EELRTFGGND TNFIDSLWQK KFSRDAYDNL QNIARILNEA

[0412] 301 KTIVGTTTPL QYMKNIFIRK YFLSDASGK ISVNKPAFKE FYRVLTRGFT ELEFVNPFKV

[0413] 361 INRKTYNLFD KAVFRINIVP DENYTINEGF NLEGANSNGQ NTEINSRNFT RLKNFTGLFE

[0414] 421 FYKLLCVRGI IPFKTKSLDE GYNKALNDLC IKVNNWDLFF SPSEDNFTND LDKVEEITAD

[0415] 481 TNIEAAEENI SSDLIQQYYL TFDFDNEPEN ISIENLSSDI IGQLEPMPNI ERFPNGKKYE

[0416] 541 LDKYTMFHYL RAQEFEHGDS RIILTNSAEE ALLKPNVAYT FFSSKYVKKI NKAVEAVIFL

[0417] 601 SWAEELVYDF TDETNEVTTM DKIADITIIV PYIGPALNIG NMVSKGEFVE AILFTGVVAL

[0418] 661 LEFIPEYSLP VFGTFAIVSY IANKVLTVQT INNALSKRNE KWDEVYKYTV TNWLAKVNTQ

[0419] 721 IDLIREKMKK ALENQAEATR AIINYQYNQY TEEEKNNINF NIDDLSSKLN RSINRAMINI

[0420] 781 NKFLDQCSVS YLMNSMIPYA VKRLKDFDAS VRDVLLKYIY DNRGTLILQV DRLKDEVNNT

[0421] 841 LSADIPFQLS KYVNDKKLLS TFTEYIKNIV NTSILSIVYK KDDLIDLSRY GAKINIGDRV

[0422] 901 YYDSIDKNQI KLINLESSTI EVILKNAIVY NSMYENFSTS FWIKIPKYFS KINLNNEYTI

[0423] 961 INCIENNSGW KVSLNYGEII WTLQDNKQNI QRVVFKYSQM VNISDYINRW MFVTITNNRL

[0424] 1021 TKSKIYINGR LIDQKPISNL GNIHASNKIM FKLDGCRDPR RYIMIKYFNLFDKELNEKEI

[0425] 1081 KDLYDSQSNP GILKDFWGNY LQYDKPYYML NLFDPNKYVD VNNIGIRGYMYLKGPRGSVM

[0426] 1141 TTNIYLNSTL YMGTKFIIKK YASGNEDNIV RNNDRVYINV VVKNKEYRLATNASQAGVEK

[0427] 1201 ILSALEIPDV GNLSQVVVMK SKDDQGIRNK CKMNLQDNNG NDIGFIGFHLYDNIAKLVAS

[0428] 1261 NWYNRQVGKA SRTFGCSWEF IPVDDGWGES SL

[0429] b. DNA sequence BoNT / A3 (SEQ ID NO: 33)

[0430] 1 atgccatttg ttaataaaca atttaattat agagatcctg taaatggtgt tgatattgct

[0431] 61 tatataaaaa ttccaaatgc aggacaaatg caaccagtaa aagcttttaa aattcatgaa

[0432] 121 ggagtatggg ttattccaga aagggatacc tttacaaatc ctgaagaagg agatttaaat

[0433] 181 ccaccaccag aagcaaaaca agttccagtc tcatattatg attcaacata tttaagtaca

[0434] 241 gataatgaaa aagataatta tttaaaggga gttataaaat tatttgacag aatttattca

[0435] 301 actgggcttg gaagaatgtt gttatcattc atagtaaagg gaataccatt ttggggtgga

[0436] 361 agtacaatag atacagaatt aaaagttatt gatactaatt gtattaatgt aatagaacca

[0437] 421 ggtggtagtt atagatcaga agagcttaat ctagtaataa caggaccctc agctgatatt

[0438] 481 atacagtttg aatgtaaaag ttttggacat gacgttttta atcttacgcg aaatggttat

[0439] 541 ggttctactc aatacattag atttagccca gattttacat ttggttttga ggagtcactt

[0440] 601 gaagttgata caatcctct tttaggtgca ggcacattg ctacagatcc agcggtaaca

[0441] 661 ttagcacatg aacttataca tgctgcacat agattatatg gatagcaat taatccaat

[0442] 721 agggtttta aagtaagac taatgcctat tatgaatga gtgggttaga agtaagcttt

[0443] 781 gaggaactta gaacatttgg gggaatgat acaacttta tagatagttt atggcaaaaa

[0444] 841 aaatttagta gagacgctta tgataatctt caaatag cacgtatact tatgaagct

[0445] 901 aaaacatag taggtactac tactccatta cagtatatga aaaatatttt tatacggaaa

[0446] 961 tattcttat ctgagatgc atctggaag atttcggtaa ataaccagc atttaggaa

[0447] 1021 ttttacaggg tgttaacaag gggtttcaca gagttagaat ttgttaatccttttaaagta

[0448] 1081 attacagga aacatattt gatttttgat aaagccgtat ttaggaataatagtaccct

[0449] 1141 gatgaaatt acacataa tgaggattt atttagaag gtgcaattctaatggtcag

[0450] 1201 aatacagaaa ttaatagtag gaattttact agattaaaaa attttacaggattatttgaa

[0451] 1261 ttttataagc tgctatgtgt aagagggata atacctttta aaactaaatcattagatgaa

[0452] 1321 ggatacaata aggcattaaa tgatttatgt atcaaagtta ataattgggacttgtttttt

[0453] 1381 agtccttcag aagataattt tactaatgat ttagataaag tagaagaaattacagctgat

[0454] 1441 accaatatag aagcagcaga agaaaatatt agttcagatt taatacaacaatattattta

[0455] 1501 acttttgatt ttgataatga acctgaaaat atttcaatag aaaatctttcaagtgatatt

[0456] 1561 ataggccaat tagagcctat gcctaatata gaaagatttc ccaatggaaaaaagtatgag

[0457] 1621 ttagataaat atactatgtt ccattatctt cgtgctcaag aatttgaacatggtgattct

[0458] 1681 agaattatct taacaaattc tgctgaagaa gcattttaa agcctaatgttgcttacaca

[0459] 1741 tttttttctt caaaatatgt aaagaagatt aataaagccg tagaggcagttatttttta

[0460] 1801 agttgggcag aagagttagt atatgacttt accgatgaaa ctaatgaagtaactactatg

[0461] 1861 gataaaattg ctgatataac tataattgtt ccatatatag ggcctgctttaataggt

[0462] 1921 aatatggtat ctaaaggaga gttcgtagaa gctatactat ttacgggagttgttgctctg

[0463] 1981 ttagaattta taccagagta ttccctccct gtatttggta cttttgcaattgtatcatat

[0464] 2041 attgccaata aggttctaac tgttcaaaca ataaataatg ctttaagtaaaagaaatgaa

[0465] 2101 aaatgggatg aagtctataa atatacagta acaaattggc tagcaaaggttaatacacag

[0466] 2161 attgatctaa tagggaaa aatgaaaaaa gctttagaaa atcaggcagaagcaacaagg

[0467] 2221 gctataataa actatcagta taatcaatat actgaggaag agaaaaataattattt

[0468] 2281 aatattgatg atttaagttc gaaacttaat aggtctataa atagggctatgattaacata

[0469] 2341 aataaattt tggatcaatg ctctgtttca tatttaatga attctatgataccttatgct

[0470] 2401 gttaaacggt tAAagattt tgatgctagt gttagagatg tattaaagtatatatat

[0471] 2461 gaatagag gaactttat tctcaagta gagatta agagagttattataca

[0472] 2521 cttagtgcag ataccttt tcagctttct aaatacgtaa atgataaaaaattattatct

[0473] 2581 acatttactg atattattaa gatatttgtt ataccctcta tattgagtatagtataaaa

[0474] 2641 aagatgatt taatagattt atctaggtat ggagcaaaa taatattggcgatagagta

[0475] 2701 tattagatt chaatagata aaatcaatt aattatta atttagaagtagtacaatt

[0476] 2761 gaggtaattt taaaaaatgc tattgtatat atagtagt atgaaattttagtactagc

[0477] 2821 ttttggaaaatttcctaa gtattttagc aagaataatc taaataatgatatacaata

[0478] 2881 aataattgta tagaaaata ttcaggatgg aaagtacac ttaattggtgaaatc

[0479] 2941 tggactttgc aggatataa gcaaacata caaagtag ttttaatacagtcaatg

[0480] 3001 gttaatatat cagattatat aaacagatgg atgtttgtaa ctatcactaataagacta

[0481] 3061 actaaatcta aaatttatat aaatggaaga ttaatagatc aaaaccaatttcaaatttg

[0482] 3121 ggtaatattc atgctagtaa taagataatg tttaaattag atggctgtagagatccacgt

[0483] 3181 agatacatca tgataaaata tttcaatctt ttcgataaag aattaaatgaaaagaaatc

[0484] 3241 aaagatttat atgatagtca atcaaatcca ggtattttaa aagactttttggggtaattat

[0485] 3301 ttacaatatg ataaaccata ctatatgtta aattatttg atccaaataaattgtcgat

[0486] 3361 gtaaataata taggtattag aggttatatg tatcttaaag ggcctagaggtagcgtaatg

[0487] 3421 actacaaaca tttatttaaa ttcaactttg tatatgggga caaaatttattaaaaaa

[0488] 3481 tatgcttctg gaaatgaaga tatattgtt agaaataatg atcgtgtatattaatgta

[0489] 3541 gtagttaaaa ataaagaata taggttagct actaatgcat cacaggcaggcgtagaaaaa

[0490] 3601

[0491] 3661 tcaaaagatg atcaaggaat aagaaataaa tgcaaaatga atttacaagataataatggg

[0492] 3721 aatgatatag gctttatagg atttcatttg tatgataata tagctaaactagtagcaagt

[0493] 3781 aattggtata atagacaagt gggaaaagct agtaggactt tcggttgttcatgggagttt

[0494] 3841 attcctgtag atgatggatg gggagaaagt tcactgtaa

[0495] c.LC / A3 coordinate, derived from crystal structure PDB:7DVL Chapter 1-18 MLD-R1 277-300 MLD-R2 300-334 MLD-R1-R2 277-334 LHD-R1-R2-R3 277-335-359 LC / A3 1-426 BoNT / A3 1292 8. BoNT / A3(V) a. Amino acid sequence of LC / A3(V) 1-426 (SEQ ID NO: 34) 1 MPFVNKPFNY RDPGNGVDIA YIKIPNAGQM QPVKAFKIHE GVWVIPERDT FTNPEEGDLN 121 STIDTELKVI DTNCINVIEP GGSYRSEELN LVITGPSADI IQFECKSFGH DVFNLTRNGY 181 GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GTFATDPAVT LAHELIHAAH RLYGIAINPN 241 RVLKVKTNAY YEMSGLEVSF EELRTFGGND TNFIDSLWQK KFSRDAYDNL QNIARILNEA 301 KTIVGTTTPL QYMKNIFIRK YFLSEDASGK ISVNKAAFKE FYRVLTRGFT ELEFVNPFKV 361 INRKTYLNFD KAVFRINIVP DENYTINEGF NLEGANSNGQ NTEINSRNFT RLKNFTGLFE421 FYKLLC a. Amino acid sequence of BoNT / A3(V) 1-1292 (SEQ ID NO: 35) 1 MPFVNKPFNY RDPGNGVDIA YIKIPNAGQM QPVKAFKIHE GVWVIPERDT FTNPEEGDLN 61 PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VIKLFDRIYS TGLGRMLLSF IVKGIPFWGG 121 STIDTELKVI DTNCINVIEP GGSYRSEELN LVITGPSADI IQFECKSFGH DVFNLTRNGY 181 GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GTFATDPAVT LAHELIHAAH RLYGIAINPN 241 RVLKVKTNAY YEMSGLEVSF EELRTFGGND TNFIDSLWQK KFSRDAYDNL QNIARILNEA 301 KTIVGTTTPL QYMKNIFIRK YFLSEDASGK ISVNKAAFKE FYRVLTRGFT ELEFVNPFKV 361 INRKTYLNFD KAVFRINIVP DENYTINEGF NLEGANSNGQ NTEINSRNFT RLKNFTGLFE 421 FYKLLLCVRGI IPFKTKSLDE GYNKALNYLC IKVNNWDLFF SPSEDNFTND LDKVEEITAD 481 TNIEAAEENI SSDLIQQYYL TFDFDNEPEN ISIENLSSDI IGQLEPMPNI ERFPNGKKYE 541 LDKYTMFHYL RAQEFEHGDS RIILTNSAEE ALLKPNVAYT FFSSKYVKKI NKAVEAVIFL 601 SWAEELVYDF TDETNEVTTM DKIADITIIV PYIGPALNIG NMVSKGEFVE AILFTGVVAL 661 LEFIPEYSLP VFGTFAIVSY IANKVLTVQT INNALSKRNE KWDEVYKYTV TNWLAKVNTQ 721 IDLIREKMKK ALENQAEATR AIINYQYNQY TEEEKNNINF NIDDLSSKLN RSINRAMINI 781 NKFLDQCSVS YLMNSMIPYA VKRLKDFDAS VRDVLLKYIY DNRGTLILQV DRLKDEVNNT 841 LSADIPFQLS KYVNDKKLLS TFTEYIKNIV NTSILSIVYK KDDLIDLSRY GAKINIGDRV 901 YYDSIDKNQI KLINLESSTI EVILKNAIVY NSMYENFSTS FWIKIPKYFS KINLNNEYTI 961 INCIENNSGW KVSLNYGEII WTLQDNKQNI QRVVFKYSQM VNISDYINRW MFVTITNNRL 1021 TKSKIYINGR LIDQKPISNL GNIHASNKIM FKLDGCRDPR RYIMIKYFNLFDKELNEKEI 1081 KDLYDSQSNP GILKDFWGNY LQYDKPYYML NLFDPNKYVD VNNIGIRGYMYLKGPRGSVM 1141 TTNIYLNSTL YMGTKFIIKK YASGNEDNIV RNNDRVYINV VVKNKEYRLATNASQAGVEK 1201 ILSALEIPDV GNLSQVVVMK SKDDQGIRNK CKMNLQDNNG NDIGFVGFHLYDNIAKLVAS 1261 NWYNRQVGKA SRTFGCSWEF IPVDDGWGES SL b. DNA sequence BoNT / A3(V) (SEQ ID NO: 36) 1 atgccctttg ttaataaacc atttaattat agagatcctg gtaatggtgt tgatattgct 61 tatataaaaa ttccaaatgc aggacaaatg caaccagtaa aagcttttaa aattcatgaa 121 ggagtatggg ttattccaga aagggatacc tttacaaatc ctgaagaagg agatttaaat 181 ccaccaccag aagcaaaaca agttccagtc tcatattatg attcaacata tttaagtaca 241 gataatgaaa aagataatta tttaaaggga gttataaaat tatttgacag aatttattca 301 actgggcttg gaagaatgtt gttatcattc atagtaaagg gaataccatt ttggggtgga 361 agtacaatag atacagaatt aaaagttatt gatactaatt gtattaatgt aatagaacca 421 ggtggtagtt atagatcaga agagcttaat ctagtaataa caggaccctc agctgatatt 481 atacagtttg aatgtaaaag ttttggacat gacgttttta atcttacgcg aaatggttat 541 gttctactc atacattag atttagccca gattttacat ttggttttga ggtcactt 601 gaagttgata caatcctct tttaggtgca ggcacattg ctacagatcc agcggtaaca 661 ttagcacatg aacttataca tgctgcacat agattatatg gatagcaat taatccaat 721 agggtttta aagtaagac taatgcctat tatgaatga gtgggttaga agtaagcttt 781 gaggaactta gaacatttgg gggaatgat acaacttta tagatagttt atggcaaaaa 841 aaatttagta gagacgctta tgataatctt caaatag cacgtatact tatgaagct 901 aaaacatag taggtactac tactccatta cagtatatga aaaatatttt tatacggaaa 961 tattcttat ctgagatgc atctggaag atttcggtaa ataaagcagc atttaggaa 1021 ttttacaggg tgttaacaag gggtttcaca gagttagaat ttgttaatccttttaaagta 1081 attachagaaaacatattt gatttttgat aaagccgtat ttaggaataatagtaccct 1141 gatgaaatt acacataa tgaggattt atttagaag gtgcaattctaatggtcag 1201 atacagaaa ttatagtag gatttttact agattaaaa attttacaggttttgaa 1261 ttttataagc tgctatgtgt aagagggata atacctttta aaactaaatcattagatgaa 1321 ggatacaata aggcattaaa ttatttatgt atcaaagtta ataattgggacttgtttttt 1381 agtccttcag aagataattt tactaatgat ttagataaag tagaagaaattacagctgat 1441 accaatatag aagcagcaga agaaaatatt agttcagatt taatacaacaatattattta 1501 acttttgatt ttgataatga acctgaaaat atttcaatag aaaatctttcaagtgatatt 1561 ataggccaat tagagcctat gcctaatata gaaagatttc ccaatggaaaaaagtatgag 1621 ttagataaat atactatgtt ccattatctt cgtgctcaag aatttgaacatggtgattct 1681 agaattatct taacaaattc tgctgaagaa gcattttaa agcctaatgttgcttacaca 1741 tttttttctt caaaatatgt aaagaagatt aataaagccg tagaggcagttatttttta 1801 agttgggcag aagagttagt atatgacttt accgatgaaa ctaatgaagtaactactatg 1861 gataaaattg ctgatataac tataattgtt ccataatatag ggcctgctttaaatataggt 1921 aatatggtat ctaaaggaga gttcgtagaa gctatactat ttacgggagttgttgctctg 1981 ttagaattta taccagagta ttccctccct gtatttgta cttttgcaattgtatcatat 2041 attgccaata aggttctaac tgttcaaca attaataatg ctttaagtaaaagaatgaa 2101 aaatgggatg aagtctataa atacagta acaattggc tagcaaggttaatacacag 2161 attgatcta taagggaaa atgaaaaaa gctttagaaa atcaggcagaagcaaagg 2221 gctataata actacagta taatxaat actgaggag agaaaatattattatttt 2281 atattgatg atttaagttc gaacttaat aggtcttaa ataggctatgatgatcata 2341 aaaattt tggatcaatg ctctgtttca tatttaatga attctatgataccttatgct 2401 gttaaacggt tAAagattt tgatgctagt gttagagatg tattaaagtatatatat 2461 gaatagag gaactttat tctcaagta gagatta agagagttattataca 2521 cttagtgcag ataccttt tcagctttct aaatacgtaa atgataaaaaattattatct 2581 acatttactg atattattaa gatatttgtt ataccctcta tattgagtatagtataaaa 2641 aagatgatt taatagattt atctaggtat ggagcaaaa taatattggcgatagagta 2701 tattagatt chaatagata aaatcaatt aattatta atttagaagtagtacaatt 2761 gaggtaattt taaaaaatgc tattgtatat atagtagt atgaaattttagtactagc 2821 ttttggaaaatttcctaa gtattttagc aagaataatc taaataatgatatacaata 2881 aataattgta tagaaaata ttcaggatgg aaagtacac ttaattggtgaaatc 2941 tggactttgc aggatataa gcaaacata caaagtag ttttaatacagtcaatg 3001 gttaatat cagattatat aacagatgg atgtttgtaa ctatcactaatatagacta 3061 actaaatcta aaatttatat aatggaga ttaatagatc aaaaccaatttcaatttg 3121 gtataatattc atgctagtaa taagataatg ttaattag atggctgtagagatccacgt 3181 agatacatca tgataaaata ttcaatctt ttcgataaag aattaatgaaaaagaatc 3241 aagatttat atgatagtca atcaatcca ggtattttaa aagactttttggggtatattat 3301 ttacaatag aaaccata ctatatgtta atttattg atccaaataatgtcgat 3361 gtaatata taggtattag aggttatatg tatcttaag ggcctagaggtagcgtaatg 3421 actacaaaca tttatttaaa ttcaactttg tatatgggga caaaatttattataaaaaaa 3481 tatgcttctg gaaatgaaga taatattgtt agaaataatg atcgtgtatatattaatgta 3541 gtagttaaaa ataaagaata taggttagct actaatgcat cacaggcaggcgtagaaaaa 3601 atactaagtg cattagaaat acctgatgta ggaaatctaa gtcaagtagtagtaatgaag 3661 tcaaaagatg atcaaggaat aagaaataaa tgcaaaatga atttacaagataataatggg 3721 aatgatatag gctttgtagg atttcatttg tatgataata tagctaaactagtagcaagt 3781 aattggtata atagacaagt gggaaaagct agtaggactt tcggttgttcatgggagttt 3841 attcctgtag atgatggatg gggagaaagt tcactgtaa c. LC / A3(V) coordinates, derived from the crystal structure PBD: 7DVL (LC / A3) N - terminus 1 - 18 MLD - R1 277 - 299 MLD - R2 299 - 334 MLD - R1 - R2 277 - 334 LHD - R1 - R2 - R3 277 - 335 - 359 LC / A3 1 - 426 BoNT / A3 1292 9. BoNT / A1 a. Amino acid sequence BoNT / A1 1-1296 (SEQ ID NO: 37) 1 MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN 61 PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG 121 STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY 181 GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKFATDPAVT LAHELIHAGH RLYGIAINPN 241 RVFKVNTNAY YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA 301 KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV 361 LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT 421 GLFEFYKLLC VRGIITSKTK SLDKGYNKAL NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE 481 ITSDTNIEAA EENISLDLIQ QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG 541 KKYELDKYTM FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA 601 AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD DFVGALIFSG 661 AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS KRNEKWDEVY KYIVTNWLAK 721 VNTQIDLIRK KMKEALENQA EATKAIINYQ YNQYTEEEKN NINFNIDDLS SKLNESINKA 781 MININKFLNQ CSVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK 841 VNNTLSTDIP FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI 901 GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP KYFNSISLNN 961 EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK YSQMINISDY INRWIFVTIT 1021 NNRLNNSKIY INGRLIDQKP ISNLGNIHAS NNIMFKLDGC RDTHRYIWIKYFNLFDKELN 1081 EKEIKDLYDN QSNSGILKDF WGDYLQYDKP YYMLNLYDPN KYVDVNNVGIRGYMYLKGPR 1141 GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKEYRLATNASQA 1201 GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFIGFHQFNNIAK 1261 LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL b. DNA sequence of BoNT / A1 (SEQ ID NO: 38) 1 ATGCCATTTG TTAATAAACA ATTTAATTAT AAAGATCCTG TAAATGGTGT TGATATTGCT 61 TATATAAAAA TTCCAAATGC AGACAAATG WIDE AAGCTTTAA AATTCATAAT 121 AAAATATGGG TTATTCCAGA AAGAGATACA TTTACAAATC CTGAAGAAGG NATIONAL 181 CCACCACCAG AAGCAAAAACA AGTTCCAGTT TCATATTATG ATTCAACATA TTTAAGTACA 241 SEQUENCES AAGATAATTA TTTAAAGGGA GTTACAAAAT CATTTGAGG ATTTTTCCA 301 ACTGATCTTG COMPETITIONGTT GTTACCATCA ATAGGG COMPETITION TTGGGGTGGA 361 CHANGE CHANGE AAAACHRINE CHANGE CHANGE CHANGE 421 CONNECTIONS SEQUENCE AGACTTAAT CTTRAGTT SequenceCCCTC AGCTGET 481 ATACAGTTTG AATGTAAAAG CTTTGGACAT GAAGTTTTGA ATCTTACGCG AAATGGTTAT 541 GGCTCTACTC AATACATTAG ATTTAGCCCA GATTTCAT TTGGTTTTGA GGAGTCACTT 601 GAAGTTGATA CAAATCCTCT TTTAGGTGCA GGCAAATTTG CTACAGATCC AGCAGTAACA 661 TTAGCATG AACTTATACA TGCTGGACAT AGATTATATG TAGCCAAT TAATCCAAAT 721 AGGGTTTTTA AAGTAAATAC TAATGCCTAT TATGAATGA GTGGGTTAGA AGTAAGCTTT 781 CONTINUOUS CONVERSATIONTTGG CONCLUSION GCAAAACC CONVERSATION ACCOUNTAAAC 841 GAATTTCGTC SEQUENCE WINDOWSTT AAAGATAG TRANSACTION WINDOW AGCT 901 AAATTAG TAG TGCTAC TGCTTCTAG TAGS AAATGTTTT TAGTAGAA 961 TATCTCCTAT CTGAAGATAC ATCTGGAAAA TTTTCGGTAG ATAAATTAAA ATTTGATAAG 1021 TTATACAAA TGTTACAGA NATIONAL TATACACA TTGTTAAGTTTTTTAAAGTA 1081 CTTAGE AAACATTT CONNECTION AAAGCCGTAT TWINTERACTATTTTACTACCT 1141 AAGGTAAATT STORM TGATGGATTT ATACAAATTTAGCAGAAAC 1201 TTTAATGGTC AAAATATACH ATTATATAT ATGATTTACT CTAAACTAAAAAATTTTACT 1261 GGATTGTTTG AATTTTATAA GTTGCTATGT GTAAGGGA TAATAACTTCTAAAACTAAA 1321 TCSEQUENCY NAGATCAA TQUALIZATION GTQTYPEATTGG 1381 QUESTION GTTTT TTAGTCCTTC AGREAT TTTACTAATG ATCTTAAAAAAAGAGATA 1441 ATTACKCTG ATTACKS FREQUENTLY ATTACKS TAGTTATTACKS 1501 SUPPORT TACCTTTAA TTTTTAX ANSWER ATATTTCTACTTACTTACT 1561 TCAAGTGACA TTATAGGCCA ATTAGAACTT ATGCCTAATA TIGAAAGATTTCCTAATGGA 1621 AAAAAGTATG AGTTAGTAA ATATACTATG TTCCATTATC TTCGTGCTCAAGAATTTGAA 1681 HELP CTAGGATTGC TTTAACAAAT TCTGTTAACG AAGCATTTATTAAATCCTAGT 1741 CGTGTTTATA CATTTTTTTC TTCAGACTAT GTAAAGAAAG TTAATAAAGCTACGGAGGCA 1801 GCTATGTTTT TAGGCTGGGT AGACAATTA GTATTATT TTACCGATGAAACTAGCGAA 1861 GTAAGTACTA CGGATAAAAT TGCGGATATA ACTATAATTA TTCCATATATAGGACCTGCT 1921 TTAAATATAG GTAATATGTT ATATAAAGAT GATTTTGTAG GTGCTTTAATTTTTTCAGGA 1981 GCTGTTATTC TGTTAGATT TATACCAGAG ATTGCAATAC CTGTATTAGGTACTTTTGCA 2041 CTTGTATCAT ATATTGCGAA TAAGGTTCTA ACCGTTCAAA CAATAGATAATGCTTTAAGT 2101 AAAAAATG AAAAATGGGA TGAGGTCTAT AAATATATAG TAACAAATTGGTTAGCAAAG 2161 GTTACK NATIONGATCT ATTACKAAAAAAAATTAG AAGCTTTACKTACKTACK 2221 GAAGCAACAA AGGCTATAAT AAACTATCAG TATAATCAAT ATACTGAGGAAGAGAAAAAT 2281 AATATTAATT TTAATATTGA TGATTTAAGT TCGAAACTTA ATGAGTCTATAAATAAAGCT 2341 ATGATTAATA TAAATAAATT TTTGAATCAA TGCTCTGTTT CATATTTAATGAATTCTTATG 2401 ATCCCTTATG GTGTTAAACG GTTAGAAGAT TTTGATGCTA GTCTTAAAGATGCATTATTA 2461 AAGTATATAT ATGATAATAG AGGAACTTTA ATTGGTCAAG TAGATAGATTAAAGATAAA 2521 GTTAATAATA CACTTAGTAC AGATATACCT TTTCAGCTTT CCAAATACGTAGATAATCAA 2581 AGATTATTAT CTACATTTAC TGAATATATT AAGAATATTA TTAATACTTCTATATTGAAT 2641 TTAAGATATG AAAGTAATCA TTTAATAGAC TTATCTAGGT ATGCATCAAAAAATAAATATT 2701 GGTAGTAAAG TAAATTTTGA TCCAATAGAT AAAAATCAAA TTCAATTATTTAATTTAGAA 2761 AGTAGTAAA TTGAGGTAAT TTTAAAAAAT GCTATTGTAT ATAATAGTATGTATGAAAAT 2821 TTTAGTACTA GCTTTTGGAT AAGAATTCCT AAGTATTTTA ACAGTATAAGTCTAAATAAT 2881 GAATATACAA TAATAAATTG TATGGAAAAT AATTCAGGAT GGAAAGTATCACTTAATTAT 2941 GGTGAATAA TCTGGACTTT ACCAGATTACT SUMMER ACAAATAATTTTTTAAA 3001 CHAPTERCAA TWOGROUND TAKEWORD GGGATTTTTGROUNDTAXCACT 3061 TALLATTACK TAAATAACTC WINDOWN CONNECTEDGGAA BETWEENCAAAAACCA 3121 ATTTCAAATT TAGTAATAT TCATGCTAGT AATATATA TGTTTAAATTAGATGGTTGT 3181 AGAGATACAC ATAGATAT TTGGATAAA TATTTTAATC TTTTTGATAAGGAATTAAAT 3241 GAAAAAGAAA CHARACTERISTICS SUPPLEMENTARY ASSISTANCE CASEGRAMMARTT 3301 TGGGGTGATT ATTTACAATA TGATAAACCA TACTATGT TAAATTTATGATCCAAAT 3361 AAATATGTCG ATGTAAATAA TGTAGGTATT AGAGGTTATA TGTATCTTAAAAGGGCCTAGA 3421 GGTAGCGTAA TGACTACAAA HELP ATTCAAGTT TGTATAGGGGGACAAAATTT 3481 ATTATAAAAA AATATGCTTC TGGAAATAAA GATAATATTG TTAGAAATAATGATCGTGTA 3541 TTATAATG TABLE AAATAAAGAA TTAGTAG CTACTAATGCATCACAGGCA 3601 GGCGTAGAAA AAATACTAAG TGCATTAGAA ATACCTGATG TAGATCTTAGAATGATCH 3661 GTAGTAATGA AGTCAAAAAA TGATCAAGGA ATAACAAATA AATGCAAAATGAATTTACAA 3721 GATAATAATG GGAATGATAT AGGCTTTATA GGATTTCATC AGTTTAATAATATAGCTAAA 3781 CTAGTAGCAA GTAATTGGTA TAATAGACAA ATAGAAAGAT CTAGTAGGACTTTGGGTTGC 3841 TCATGGGAAT TTATTCCTGT AGATGATGGA TGGGGAGAAA GGCCACTGTA A

[0496] Example 5

[0497] Botulinum toxin type A1 (BoNT / A1) is one of the most potent human protein toxins, but also an important therapeutic agent. BoNT / A1 contains a catalytically active light chain (LC / A1) linked to the heavy chain via disulfide bonds. How intracellular LC / A1 localizes to the plasma membrane to cleave synaptosome-associated protein 25 (SNAP-25) remains unclear. Imaging analysis revealed that LC / A1 migrates along microtubules and co-localizes with several Rab GTPases. Using a variety of dominant-negative (DN) Rab GTPases, we demonstrated for the first time that LC / A1 utilizes a rapid synaptic vesicle reuptake pathway to transport and cleave SNAP-25 bound to the plasma membrane. Mechanistic studies indicate that the stable intracellular localization of the light chain determines the potency and duration of action of BoNT / A1. These results open the possibility of custom-engineering novel BoNT therapies and identifying new host protein targets that limit BoNT toxicity.

[0498] Botulinum toxin (BoNT) is a Category A 1 controlled substance and is considered a potential biological weapon due to its high toxicity and the severity and long duration of botulism in humans and vertebrates (1). There is currently no approved human botulism vaccine, and the therapeutic window for heptavalent equine antiserum (BAT) is limited; it is only effective in the early stages of the disease, before BoNT enters motor neurons. Once inside neurons, BoNT persists and cleaves membrane-bound v- or t-SNARE proteins, resulting in the persistent (weeks to months) flaccid paralysis characteristic of botulism (1). Every botulism outbreak is a legally reportable public health emergency (2).

[0499] BoNTs are a structurally conserved family of proteins containing a catalytically active light chain (LC, zinc metalloproteinase) linked to a heavy chain (HC) via disulfide bonds. HC mediates binding to host cell receptors and LC translocation into the cytoplasm of motor neurons. BoNTs comprise seven immunologically distinct serotypes (AG) and more than 40 subtypes, of which BoNT / A comprises eight subtypes (3). The BoNT / A1 subtype is an effective and widely used human therapy for a variety of neuromuscular diseases (4). Although the entry of BoNT / A1 and the translocation of LC / A1 into the neuronal cytoplasm have been studied, the molecular mechanism by which LC / A1 is transported intracellularly to the plasma membrane to cleave the 25-kDa membrane-bound t-SNARE synaptosome-associated protein (SNAP-25) remains unresolved. In general, how the catalytic components of protein toxins target their intracellular sites remains poorly understood.

[0500] Identification of the BoNT / A subtypes revealed that BoNT / A3 has a shorter duration of action, a characteristic of its LC (1). LC / A3 is localized to intracellular vesicles, while LC / A1 is stably localized to the plasma membrane of the mouse neuroblastoma cell line Neuro-2A (N2A) via two steps (5). The N-terminus of LC / A1 (called A1N, residues 1-17) mediates the binding of LC to intercellular vesicles, while the internally encoded membrane localization domain (called MLD, residues 275-334) mediates the stable localization of LC / A1 on the plasma membrane (5–8). In a mouse model of botulism, LC-mediated vesicle association and stable plasma membrane association contribute to the high potency of BoNT / A1 (9).

[0501] Targeted intracellular vesicle transport is facilitated by vesicle-bound Rab GTPases, members of the Ras superfamily of small G proteins (10). Therefore, Rab GTPases can serve as molecular markers for vesicle transport pathways (11). However, the intracellular functions and localizations of Rab GTPases often overlap within neuronal terminals, necessitating the identification of multiple Rab GTPases to elucidate intracellular transport pathways (12). By using subsets of dominant-negative (DN) Rab GTPases and the LC / A1 and LC / A3 isoforms, LC / A1 was found to transport to the plasma membrane via a rapid branch of the synaptic vesicle reuptake pathway and cleave SNAP-25. The membrane-stabilizing association of LC / A1 is related to its high potency and long-lasting effect.

[0502] Microtubule-mediated intracellular transport of LC / A1 in N2A cells

[0503] Microtubules and actin are key components of intracellular motility, including cytokinesis, transport, and locomotion, and some microbial pathogens utilize microtubules and actin for intracellular transport (13, 14). Two cytoskeleton inhibitors were tested to investigate the role of actin and / or microtubules in the intracellular localization of LC / A1 in N2A cells: cytochalasin D (binds to the polymeric ends of actin filaments to disrupt actin elongation) (15, 16); and nocodazole (binds to β-tubulin, leading to microtubule depolymerization) (17). N2A cells were transfected overnight using plasmids encoding EGFP-LC / A1 (membrane localizer) or EGFP-LC / A3 (vesicle localizer). Cells were then treated with cytochalasin D or nocodazole, and actin (phalloidin) and microtubules (β-tubulin) were stained (15, 16). Treatment of N2A cells with cytochalasin D resulted in actin disruption and the formation of neuronal buds containing microtubules (β-tubulin-positive filaments). In these cells, LC / A1 is retained on the plasma membrane, while LC / A3 is retained in intracellular vesicles. Figure 15A Therefore, actin disruption did not alter the localization of ectopically expressed LC / A1 on the plasma membrane or the vesicle association of LC / A3. Real-time fluorescence imaging of CD-treated N2A cells showed membrane-localized LC / A1 and its bidirectional transport in neuronal buds (data not shown). In contrast, in nocodazole-treated N2A cells, ectopically expressed LC / A1 remained on the plasma membrane, while LC / A3 translocated from intracellular vesicles to the cytoplasm. Figure 15A In summary, these data suggest that microtubules facilitate the association of LC / A with intracellular vesicles and their intracellular transport to the plasma membrane.

[0504] In N2A cells, LC / A1 co-localizes with Rab GTPase.

[0505] The above results indicate that LC / A utilizes microtubules for intracellular transport. Given the complexity of the neuronal vesicle pathway (11), a group of Rab GTPases (18) that contribute to vesicle compartmentalization and play a role in synaptic vesicle migration and / or circulation were used to investigate intracellular transport of LC / A1. Figure 15B At steady state, LC / A1 showed the highest mean Pearson colocalization coefficient (PCC) with the fast-circulating endosome marker Rab4 and the late synaptic vesicle (SV) marker Rab27a (19). LC / A1 showed moderate PCC with Rab5 (clathrin-dependent endocytosis marker, also essential for SV formation (11, 20)), Rab11 (slow-circulating and early sorting endosome marker (19)), and Rab7 (late endosome marker (21)). LC / A1 showed low colocalization with Rab3a (late SV marker (19)). Figure 15CAnalysis of intracellular compartments in individual cells showed that in N2A cells, LC / A1 co-localized along the plasma membrane with Rab3a, Rab27a, and Rab4, co-localized intracellularly with Rab5 and Rab11, and co-localized with Rab7 in the perinuclear region. Figure 15B In summary, under steady state, LC / A1 colocalizes with Rab GTPases involved in a variety of intracellular functions, including circulating endosomes, synaptic vesicle circulation, and retrograde transport endosomes (22).

[0506] Dominant-negative (DN) Rab GTPases inhibit N-terminal LC / A1-mediated stable plasma membrane localization.

[0507] DN Rab GTPases block Rho guanylate exchange factor (GEF), interfering with the exchange of nucleotides by Rab GTPases and thus preventing Rab GTPase-directed vesicle movement (12, 23). Vesicle interactions mediating the intracellular transport of LC / A1 to the plasma membrane were identified by co-transfecting N2A cells with DNA encoding DsRed-LC / A1 and individual EGFP-DN Rab GTPases (23, 24), where co-expression with DN Rab3a, DN Rab4, DN Rab5, and DN Rab27b inhibited LC / A1 plasma membrane localization. Figure 16A However, co-expression with either DN Rab7 or Rab11 did not alter the plasma membrane localization of LC / A1, indicating that neither Rab7 nor Rab11 alone is essential for LC / A1 membrane localization. The observed inhibitory effects of DN Rab3a, DN Rab4, DN Rab5, and DN Rab27b on LC / A1 transport suggest that LC / A1 transport to the plasma membrane utilizes the rapid SV cycle pathway. LC / A3V (A1-MLD) (cytoplasmic LC / A3 chimera, showing inefficient plasma membrane localization independent of intracellular vesicles (9)) was tested to determine whether LC-vesicle association is required to observe the inhibitory effect of DN Rab GTPases on LC / A1 transport to the plasma membrane. Figure 16B The efficiency of LC / A3V (A1-MLD) localization to the plasma membrane was not inhibited by co-expression of DN Rab3a, DN Rab4, DN Rab5, or DN Rab27b, consistent with the membrane localization of LC / A3V (A1-MLD) independent of intracellular vesicles (9). These data also support the role of the vesicle-mediated rapid SV circulation pathway in LC / A1 transport to the plasma membrane. The intracellular transport of cholera toxin B subunit (CT / B) and transferrin in N2A cells was assessed by co-expression with individual DN Rab GTPases, validating the specificity of DN Rab GTPase function (25,26). Figure 18Co-expression of DN Rab4 or DN Rab11 had limited effects on transferrin uptake and circulation, and endocytosis of CT / B was not inhibited by DN Rab3a, DN Rab4, DN Rab7, DN Rab11, or DN Rab27b, which function downstream of endocytosis (11). Overall, these experiments suggest that the N-terminus of LC / A1 plays a role in the transport of LC / A1 to the plasma membrane via the rapid SV circulation pathway.

[0508] DN Rab GTPase inhibits N-terminal LC / A1-mediated cleavage of membrane-bound SNAP-25.

[0509] Although the inhibition of LC / A1 plasma membrane localization by DN Rab GTPase defined the overall intracellular transport of LC / A1, subsequent experiments tested the ability of DN Rab GTPase to inhibit LC / A1 SNAP-25 cleavage. N2A transfection conditions for pEGFP-LC / A1 were determined to achieve an intracellular dose of LC / A1 capable of cleaving approximately 30% of the total intracellular SNAP-25, as detected by Western blotting. Figure 19A -B). Imaging of individual cells transfected solely with pEGFP-LC / A1 showed that LC / A1 expression ranged from 1x10⁻¹. 7 Up to 6x10 7 The total fluorescence intensity is proportional to the amount of SNAP-25 cleaved. Figure 19E pEGFP-LC / A1 was co-transfected with an individual plasmid encoding DN Rab GTPase (expression was confirmed by Western blotting). Figure 19C The results showed that LC / A1 cleavage of SNAP-25 was significantly inhibited by co-expression of DN Rab27b, DN Rab5, DN Rab3a, or DN Rab4, while no significant inhibition of SNAP-25 cleavage by LC / A1 was observed by co-expression of DN Rab7 or DN Rab11. Figure 19E The cleavage of SNAP-25 by LC / A3V (A1-MLD) (a cytoplasmic LC / A3 chimera that is independent of intracellular vesicle localization to the plasma membrane) is not inhibited by co-expression of DN Rab27b, DN Rab5, DN Rab3a, or DN Rab4. Figure 19F -G). Overall, these data are consistent with the association between LC / A1 and membrane stability through rapid synaptic vesicle recovery pathways for efficient SNAP-25 cleavage.

[0510] Intracellular stability determines the potency and duration of action of LC / A.

[0511] Typical intracellular eukaryotic proteins have a mean half-life ranging from 1.5 to 48 hours (27–29), however, BoNT / LC can persist in motor neurons for weeks to months and continuously cleave SNARE proteins (1, 30). The retention of SNAP-25 cleavage can be used to measure the duration of BoNT / A action in cultured cells (1); among these, BoNT / A1 has a longer duration of action than vesicle-bound BoNT / A3 (5), and BoNT / A1-A3 chimeras suggest that LC determines the duration of BoNT action (8). To elucidate the basis of LC / A potency and the duration of BoNT action, researchers developed two novel cell-based assays.

[0512] Indicating the decay of intracellular LC / A in motor neurons by measuring the re-emergence of uncut SNAP-25 (1), motor neurons transfected with equimolar amounts of mRNA encoding membrane-localized LC / A1, vesicle-associated LC / A3, and cytoplasmic LC / A3V showed that, over 22 days, the re-emergence rate of uncut SNAP-25 in LC / A1 and LC / A3 was slower than that in LC / A3V (1). Figure 20A This suggests that the stability of cytoplasmic LC / A3V is less than that of membrane-localized LC / A1 and vesicle-associated LC / A3. In motor neuron mRNA transfection experiments, using mRNAs encoding LC / A1-A3MLD and LC / A3-A1MLD and monitoring SNAP-25 cleavage for 72 days, the results showed that the A isotype of MLD (previously shown to determine the membrane localization of LC / A1) plays an important role in LC stability. Figure 20B This is the first time that intracellular localization has been shown to determine the duration of LC / A action.

[0513] Next, the evaluation of intracellular LC / A expression and function in N2A cells provided evidence for the efficacy mechanism of intracellular LC / A in SNAP-25 cleavage. Preliminary experiments showed that overnight transfection of N2A cells with serially diluted pEGFP LC / A (A1, A3, or A3V) allowed for monitoring of intracellular SNAP-25 cleavage and LC expression (via GFP signaling), thus achieving dose-dependent intracellular efficacy measurement. Figure 20C In in vitro assays, earlier studies showed that LC / A3 cleaved SNAP-25 at a rate similar to LC / A1 (31). However, when transfected with an equimolar amount of pEGFP-LC, intracellular LC / A3 showed approximately 3 times greater SNAP-25 cleavage capacity than LC / A1 (31). Figure 20D -E), approximately 30 times stronger than LC / A3(V). Figure 20D-E). Under conditions of equal plasmid transfection, the steady-state expression of LC / A3V was statistically lower than that of LC / A3 or LC / A1. To assess the basis of the lower steady-state expression, pEGFP-LC / A3V (A1 N), pEGFP-LC / A3V (A1 MLD), and pEGFP-LC / A3V (A1 N, MLD) chimeras were synthesized using pEGFP-LC / A3V as a platform. Serial dilutions of N2A cells transfected with the LC / A3(V)-A1 chimera showed that protein expression levels and SNAP-25 cleavage efficacy were correlated with whether MLD originated from A1 or A3. Figure 20F ).

[0514] Evaluation of full-length BoNT / A3V showed that the N-terminal composition modulated the potency of BoNT; in a botulism-induced mouse model, BoNT / A3V was approximately 5-fold less potent than BoNT / A3, and also had lower rotator test and DAS scores. Figure 20G -H). BoNT / A3V exhibits a slightly shorter duration of action in vivo compared to BoNT / A3, suggesting a greater difference at equimolar doses, as mice required an injection of five times the total amount of BoNT / A3V protein to observe paralysis. Therefore, the actual difference in duration is greater than implied by in vivo studies, and the biological characteristics of full-length BoNT / A3V correlate with the cellular characteristics of LC / A3V. Overall, these studies indicate that the N-terminus and MLD mediate stable membrane associations of LC / A1, thereby enabling the long-lasting action of BoNT / A1.

[0515] discuss

[0516] This is the first study to map the intracellular transport pathway of LC / A1 to the plasma membrane to cleave SNAP-25 and to define the intracellular dynamics of LC / A1 via the rapid synaptic vesicle signaling pathway. Previous studies have shown that intracellular LC / A1 is stably located on the plasma membrane, while LC / A3 is mainly located in vesicles and cytoplasm, and undergoes dynamic import and export transport across the plasma membrane, while the LC / A3V variant is mainly found in the cytoplasm (5). The microtubule disruptor nocodazole can displace LC / A3 located in vesicles into the cytoplasm of N2A cells, but does not affect LC / A1 located on the membrane (5). Figure 15A ).

[0517] Therefore, microtubules are essential for the dynamic intracellular transport of LC / A to the plasma membrane. BoNT entry into neurons is a multi-step process; prior to endocytosis, BoNT / A1 binds to the ganglioside / synaptic binding protein 1 complex and synaptic vesicle protein SV2 on the neuronal surface (32,33). BoNTs primarily enter the cell via a clathrin-dependent pathway in an intracavitary form. 34Studies on BoNT-HC entry into N2A cells showed that nocodazole treatment reduced HC binding, while genistein (an inhibitor of pit-mediated endocytosis and the actin network) did not reduce HC binding. 35 , 36 Our intracellular transport data complement these studies, demonstrating that BoNT / A utilizes microtubules for entry and intracellular transport. This data is further reinforced by the observation of bidirectional movement of LC / A1 in CD-treated N2A cell neuronal buds (data not shown), consistent with a previous study showing that CD-treated cultured neurons remain sensitive to BoNT / A cleavage of SNAP-25 (37).

[0518] Early endosome formation required for synaptic vesicle circulation is mediated by Rab5, which co-localizes with LC / A1. Consistently, DNRab5 inhibits the migration of LC / A1 to the plasma membrane, retaining LC / A1 on intracellular vesicles. Figure 16A (20). Rab5(ac)( 42 Clathrin-mediated endocytosis is the main pathway for the recovery of synaptic vesicles from the plasma membrane. 11 , 34 , 43 Previously, Rab5 has been shown to be involved in the transport of EGF (44) and ricin (45), but not in the endocytosis of Shiga toxin (44), and in Yersinia ( Yersinia ADP ribotransferase (from Clostridium difficile) C. difficile The targeting effect of glycosylated toxins (46). In Drosophila, DN Rab5 mediates isomorphic fusion of synaptic vesicles, increases synaptic vesicle size and inhibits neurotransmitter release ( 47 Furthermore, DN Rab5 inhibits early endosome fusion (…). 48 Under steady-state conditions, co-expression of DNRab5 statistically inhibited the membrane localization of LC / A1. Figure 16A ) and the cutting of SNAP-25 for membrane positioning ( Figure 19D The presence of -E indicates that DN Rab5 blocks a necessary step in the LC / A1 transport pathway to the plasma membrane. The inhibition of intracellular transport and SNAP-25 cleavage by DN Rab5 supports the idea that early endosomes are involved in LC / A1 transport, occurring before Rab4-positive late endosome formation, which is also consistent with the co-localization of Rab5 and LC / A1 in the cytoplasm. Figure 15B ).

[0519] Both Rab4 and Rab11 promote endosomal resorption prior to SV formation. 19Therefore, kinin-2 mediated endosome recycling was divided into two groups: Rab4-mediated rapid recycling and Rab11-mediated slow recycling (19, 49, 50). Ricin has been reported to be sensitive to DN-Rab4 (45). Although both Rab4 and Rab11 are present on the recycling endosome, LC / A1 shows greater co-localization with Rab4 than with Rab11 ( ). Figure 15B Furthermore, DN Rab4 (but not DN Rab11) inhibited the transport of LC / A1 to the plasma membrane. Figure 16A Furthermore, DN Rab4 is more efficient than DNRab11 in inhibiting LC / A1 cleavage of SNAP-25. Figure 19E This indicates that LC / A1 preferentially utilizes the Rab4-mediated rapid synaptic vesicle reuptake pathway to reach the plasma membrane. In neurons, Rab3a and Rab27b control synaptic vesicle docking, fusion, and exocytosis. 19 Rab3(ad) ( 51 It is crucial for kinin-1 mediated rapid anterograde migration and vesicle assembly on microtubules. 52 Although Rab3b, 3c, and 3d have functional redundancy, knockout models show that removal of Rab3a leads to postnatal death (53). Rab3 and Rab27(ab) (54) are structurally related, partially co-localized, and share effector proteins such as Rabphilin3a, which binds to Rab3 and preferentially to Rab27, regulating the SV cycle before transport and localization to the plasma membrane with SNAP-25 (39,40). Co-localization of LC / A1 with Rab27a was observed to be higher than that with Rab3a ( Figure 15C Furthermore, co-expression of DN Rab3a or DN Rab27b inhibited the transport of LC / A1 to the plasma membrane. Figure 16A ). Inhibition of LC / A1 cleavage of SNAP-25 by DN Rab3a or DN Rab27b ( Figure 19E Consistent with previous studies, this research indicates that Rab3 is essential for SNARE complex assembly and cellular sensitivity to BoNT (55). Although present in overlapping libraries, Rab3 and Rab27 transition to different SVs over time (39), which could explain why expression of either DN Rab3a or DN Rab27b inhibits LC / A1 translocation to the plasma membrane, but shows greater co-localization with Rab27a. Overall, these data suggest that late synaptic vesicle circulation is a step in LC / A1 membrane localization and SNAP-25 cleavage. Although DN Rab7 does not interfere with LC / A1 translocation, localization, or SNAP-25 cleavage at the plasma membrane ( Figure 16A , Figure 19EHowever, Rab7 is essential for the effective poisoning and sorting of the Shiga toxin B subunit (56). LC / A1, along with Rab7 and other Rab GTPases involved in retrograde protein transport (such as Rab5), Figure 15C The co-localization of LC / A1 may reflect the co-interaction between LC / A1 and intracellular vesicles involved in both anterograde and retrograde SV regeneration pathways (delivering LC / A1 to the plasma membrane), as observed in LC / A3V (A1MLD). Figure 19G This aligns with a wealth of literature supporting retrograde and anterograde transport of BoNT / A1 in motor neurons (5, 6, 9, 57). In N2A models lacking long axonal and neurite connections in motor neurons, these pathways are not expected to be essential for LC / A1 function and membrane localization. However, we propose that in motor neurons, LC is distributed intraneurally to reach all synapses using a Rab5- and Rab7-dependent axonal transport system. In summary, these data demonstrate that LC / A1 utilizes circulating synaptic vesicles for transport to the plasma membrane.

[0520] In previous studies, we identified the N-terminal 17 amino acids (N-terminus) and the approximately 75-amino acid motif (MLD) of LC / A1 as crucial for its membrane localization; LC / A3 has an altered MLD, leading to its vesicle localization; and LC / A3V additionally includes an altered N-terminus, resulting in its cytoplasmic localization (9). The disruption of LC / A3 vesicle association by the microtubule inhibitor nocodazole, and the finding that the membrane localization of the LC / A3V (A1-MLD) chimera is unaffected by the DN Rab enzyme of the rapid synaptic vesicle circulation pathway, together suggest that the N-terminus is involved in vesicle association and intracellular transport. Using a novel mRNA-based cellular assay to investigate the duration of action of intracellular LC, we further demonstrated that both the N-terminus and MLD play a role in determining the duration of action ( Figure 20A ,B); and animal studies have demonstrated the role of the N-terminus in determining potency. Figure 20G In summary, these data confirm the correlation between intracellular localization and potency and duration of action, involving the combined contributions of the N-terminus (associated with intracellular transport) and the MLD (previously shown to be associated with stable membrane localization of LC / A1) (9).

[0521] Our findings identify novel potential host proteins for BoNT therapy (11), including Rabip4 (a Rab4 effector involved in controlling early endosome transport from reclaimed endosomes to sorted endosomes) and Rabphillin3A (38) (a Rab3 / Rab27 effector that can bind to the N-peptide of SNAP-25 to promote the assembly of the SNARE complex during exocytosis (39–41)).

[0522] Based on our results, and combined with previous data on Rab GTPase activity (11,26) and BoNT transport and LC / A localization (5,35,57), we propose an intracellular LC / A1 transport model. Figure 21 LC / A1 binds to Rab11 / Rab4-positive recycling endosomes, a process mediated by the N-terminal 17 amino acids (5). Subsequently, LC / A1 preferentially utilizes rapid Rab4-positive recycling endosomes; after the recycling endosomes have completed initiation and loaded neurotransmitters, LC / A1 translocates to Rab3 or Rab27-positive synaptic vesicles. Upon approaching the plasma membrane, the membrane-localizing domain of LC / A1 (residues 275-334) binds and assists in the cleavage of SNAP-25, making LC / A1 a stable membrane-localizing protein, where it subsequently continues to cleave SNAP-25 (9). An essential step in LC / A1 transport is the formation of early Rab5 endosomes, which is crucial for the formation of downstream vesicles such as Rab4-positive recycling endosomes. Rab5 and Rab7 are also involved in protein degradation via phagosomes, so colocalization with these Rab enzymes may suggest a role for phagosomes / autophagosomes in the slow degradation of LC / A1.

[0523] Materials and methods in Example 5

[0524] Unless otherwise stated, all reagents were purchased from Life Technologies (Grand Island, NY, USA).

[0525] Engineering of the EGFP-LC / A construct and Rab dominant-negative variants 5

[0526] As mentioned earlier, plasmid pEGFP-C3, encoding DNA from A1 (1-450) and A3LM (1-446), was engineered. 5 The enhanced green fluorescent protein (EGFP) fusions of LC / A1, LC / A3LM, and Rab GTPases 3a, 4, 5, 7, 11, and 27a were subcloned into the SacI-BamHI restriction site of pEGFP-C3. Primers were designed and obtained from New England Biolabs® NEBaseChanger® (Ipswich, Massachusetts, USA) to the engineered dominant-negative (DN) variant of the Rab protein: Rab3a (T 36 N), Rab4 (S) 22 N), Rab5 (S) 34 N), Rab7 (T) 22 N), Rab11(S) 25 N) and Rab27b (T 23 N).

[0527] Cell culture

[0528] Neuro-2A (N2A) cells were derived from ATCC (Manassas, Virginia, USA) CCL-131. N2A cells were cultured in a complete essential medium supplemented with 10% fetal bovine serum, 1x penicillin-streptomycin, 0.1% sodium bicarbonate, 1 mM sodium pyruvate, and 1% non-essential amino acids at 37°C in a humidified 5% CO2 (v / v) environment.

[0529] Human motor neurons were cultured and prepared according to methods known in the art and the above description.

[0530] N2A cell transfection and cytoskeleton inhibitor treatment

[0531] As described above, N2A cells were transfected ( 58 N2A cells were seeded at a density of 50,000 cells / well in 24-well plates on acid-etched glass coverslips coated with poly-D-lysine (1:500).

[0532] Cytoskeleton inhibitors: The following day, as previously described, N2A cells were transfected with 500 ng of the specified plasmid (Lipofectamine LTX; Invitrogen). ™ (Waltham, Massachusetts, USA) 5 After overnight transfection, cells were treated for 60 minutes with either 4 µM cytochalasin D (C8273, Sigma-Aldrich, St. Louis, USA) or 9 µM nocodazole (M1404, Sigma-Aldrich, St. Louis, USA).

[0533] DN Rab GTPase co-localization: The following day, as previously described, N2A cells were transfected with 500 ng pEGFP-LC / A1 (Lipofectamine LTX; Invitrogen). ™ (Waltham, Massachusetts, USA) 5 ).

[0534] DsRed-A1 or A3V (A1 MLD) and EGFP-DN Rab GTPase: The next day, as described above (5), N2A cells were transfected with the specified plasmid using 2000 ng pDsRed-LC / A1 or pDsRed-LC / A3V (A1MLD) and 500 ng pEGFP-DNRab GTPase (Lipofectamine LTX; Invitrogen). ™ (Waltham, Massachusetts, USA) 5 ).

[0535] DN Rab GTPase cleavage of SNAP-25: The following day, as previously described, N2A cells were transfected with 31.25 ng pEGFP-LC / A1, 125 ng pEGFP-DN Rab GTPase, and pEGFP, bringing the total plasmid dose to 656.25 ng (Lipofectamine LTX; Invitrogen). ™ (Waltham, Massachusetts, USA) 5 ).

[0536] Immunofluorescence imaging (IF)

[0537] After overnight transfection, N2A cells were fixed with 4% (w / v) paraformaldehyde for 15 minutes at room temperature. The cells were then washed twice with room temperature PBS and distilled into DBPS (14040182, Gibco) containing 10% FBS (v / v), 2.5% cold-water fish skin gelatin (w / v), 0.1% Triton-X (v / v), and 0.05% Tween-20 (v / v). ™ (In Illinois, USA) 60 minutes closed. Unless otherwise stated.

[0538] Cytoskeleton or Rab: N2A cells were incubated with primary antibodies: 1:5000 rabbit α-β-3 tubulin (ab52623, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab3a (ab3335, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab4 (ab109009, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab5 (ab218624, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab7 (ab137029, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab11 (ab180504, Abcam, Cambridge, UK) or 1:1000 rabbit α-Rab27a (ab223044, Abcam, Cambridge, UK), in a solution containing 5% FBS (v / v), 1% cold-water fish skin gelatin (w / v), and 0.1%... Incubate overnight at 4°C with shaking in DBPS (incubation solution) containing Triton-X (v / v) and 0.05% Tween-20 (v / v). The next day, N2A cells were washed three times (5 min each) with DPBS containing 0.005% Tween-20 (v / v) and incubated for 60 min at room temperature with Alexa-Fluor-conjugated secondary antibody or phalloidin-Alexa-fluor 568 nm (1:250) (A12380, Life Technologies, Wisconsin, USA). Cells were then washed twice with 0.005% Tween-20 (v / v) DPBS and incubated with Hoechst (1:10,000 in DPBS, H21492, Invitrogen). ™ Stain for 5 minutes using a microscope slide (Invitrogen, Massachusetts, USA). Flip the coverslip onto the microscope slide and cure with 8 µL Prolong™ Gold anti-fluorescence quenching mounting medium (Invitrogen, Massachusetts, USA).

[0539] GFP and DNA colocalization: After overnight transfection, N2A cells were washed twice with DPBS at 4°C and stained with wheat germ lectin (WGA) (1:1000, W32466, Invitrogen, Massachusetts, USA) as a membrane dye at 4°C for 30 minutes. After WGA incubation, N2A cells were washed twice with DPBS, then fixed with 4% (w / v) paraformaldehyde at room temperature for 15 minutes, washed twice with DPBS, and then stained with Hoechst (1:10,000 in DPBS, H21492, Invitrogen). ™Treat with a nuclear marker (Invitrogen, Waltham, Massachusetts, USA) at room temperature for 5 minutes. Flip the coverslip onto the microscope slide and cure with 8 µL Prolong™ Gold anti-fluorescence quenching mounting medium (Invitrogen, Waltham, Massachusetts, USA).

[0540] SNAP-25 cleavage assay: N2A cells were incubated with primary antibody: 1:1500 mouse anti-SNAP-25 (ABIN236419, Antibodies-Online (Limerick, Pennsylvania, USA)) in DBPS (incubation solution) containing 5% FBS (v / v), 1% cold-water fish skin gelatin (w / v), 0.1% Triton-X (v / v), and 0.05% Tween-20 (v / v) overnight at 4°C with shaking. The following day, N2A cells were washed three times (5 minutes each) with DPBS containing 0.005% Tween-20 (v / v) and incubated for 60 minutes at room temperature with Alexa-Fluor-conjugated secondary antibody or phalloidin-Alexa-fluor 568 nm (1:1000) (A12380, Life Technologies, Wisconsin, USA). The cells were then washed twice with 0.005% Tween-20 (v / v) DPBS and incubated with Hoechst (1:10,000 in DPBS, H21492, Invitrogen). ™ Stain for 5 minutes using a microscope slide (Invitrogen, Massachusetts, USA). Flip the coverslip onto the microscope slide and cure with 8 µL Prolong™ Gold anti-fluorescence quenching mounting medium (Invitrogen, Massachusetts, USA).

[0541] Cholera toxin-B subunit and transferrin: After overnight transfection with DN Rab GTPases (3a, 4, 5, 7, 11, or 27b), N2A cells were washed twice with DPBS at 4°C and treated for 5 min at 37°C and 5% CO2 with either 200 mM cholera toxin-B subunit-AlexaFluor 594 conjugate (C34777, Thermo, Illinois, USA) or 200 mM transferrin-AlexaFluor 594 conjugate (T13343, Thermo, Illinois, USA). N2A cells were washed twice with DPBS, then fixed with 4% (w / v) paraformaldehyde at room temperature for 15 min, washed twice with DPBS, and treated with Hoechst (1:10,000 in DPBS, H21492, Invitrogen). ™The sample (from Waltham, Massachusetts, USA) was treated as a nuclear marker at room temperature for 5 minutes. The coverslip was then flipped onto a microscope slide and cured with 8 µL Prolong™ Gold anti-fluorescence quenching mounting medium (Invitrogen, Waltham, Massachusetts, USA).

[0542] Immunofluorescence imaging

[0543] As previously described, N2A cells were cultured and analyzed ( 5 , 58 ).

[0544] Fixed cell imaging: N2A cells were imaged using a Nikon Eclipse Ti inverted microscope with a 60x oil immersion objective (1.4 NA objective), and the data were analyzed using Eclipse software.

[0545] Live-cell imaging: As described above, N2A cells were treated with 4 µM cytochalasin D and incubated for 60 minutes seven hours after LC / A1 transfection. Live-cell imaging of N2A cells was performed using a 60x oil immersion objective (1.4NA objective) on a Nikon Eclipse Ti inverted microscope with the stage heated to 37°C (Frank E. Fryer A-50). Data analysis was performed using Eclipse software. Images were acquired every 15 seconds for 15 minutes. The video was compiled using Nikon Elements AR 1.60.00 64-bit software (Melville, NY, USA) and ImageJ. 59 , 60 ).

[0546] Western Imprint

[0547] EGFP-TRAP®_MA elution buffer was collected in 2x PBS and subjected to SDS-PAGE gel electrophoresis as described above. 5Samples were separated on 13.5% SDS-PAGE and transferred to an Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, Massachusetts, USA). The PVDF membrane was rehydrated in methanol, washed with reverse osmosis water, stained with Ponceau S (P3504, Sigma-Aldrich, St. Louis, USA), and incubated in blocking buffer (2% milk powder (w / v) in 0.1% TBST). The PVDF membrane was incubated overnight at 4°C with shaking in 1:4000 or 25,000 rat anti-EGFP monoclonal IgG (3H9, Chromotek, Prannegre, Germany) and 1:4000 mouse anti-SNAP-25 monoclonal IgG (111011, SYSY, Göttingen, Germany). The bound primary antibody was recognized by horseradish peroxidase-conjugated secondary antibody at a dilution of 1:10,000 against rat or 1:10,000 against mouse (Life Technologies, SMASS). The secondary antibody was imaged using a Super Signal™ West Pico PLUS chemiluminescent substrate (34578, Thermo, Illinois) on an Azure C600 imaging system (Dublin, CA) with a 60-second exposure.

[0548] References for Example 5

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[0609] As will be apparent to those skilled in the art from the foregoing description, various substitutions and modifications can be made to the invention disclosed herein without departing from its scope and spirit. The inventions exemplarily described herein can be suitably practiced in the absence of any one or more elements or limitations not specifically disclosed herein. The terms and expressions used herein are for illustrative purposes and not for limiting purposes; their use does not exclude any equivalent features or portions thereof shown and described, and it should be recognized that various modifications are likely to fall within the scope of the invention. Therefore, it should be understood that although the invention has been described through specific embodiments and optional features, those skilled in the art can modify and / or alter the concepts disclosed herein, and such modifications and alterations should be considered within the scope of the invention. Specifically, any clostridium neurotoxin (or other SNARE-cleaving homolog) light chain mRNA or a truncated or extended version thereof may be used for therapeutic purposes and is considered to be within the scope of the invention. Additional mRNA production or delivery methods can and are contemplated to be applied to deliver mRNA to target cells in vivo, and therapeutic use using mRNA encoding SNARE-cleaving enzymes to treat diseases related to exocytosis is considered to be within the scope of the invention, regardless of the delivery carrier or mRNA design.

[0610] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all embodiments / examples herein is merely for the purpose of better illustrating the invention and not for limiting the scope of the invention as claimed, unless otherwise indicated. All language in this specification should not be construed as indicating non-claimed elements necessary for the practice of this invention.

[0611] This document cites numerous patent and non-patent references. All cited references are incorporated herein by reference in their entirety. If a term's definition in this specification differs from its definition in any of the cited references, the definition in this specification shall prevail.

Claims

1. A method comprising: The synthesized mRNA construct is given to the recipient to produce clostridial neurotoxin (BoNT) or SNARE-cleaving homolog light chain or a variant thereof.

2. The method of claim 1, wherein the BoNT or SNARE cleavage homologous light chain is a light chain of BoNT / A1 or a variant thereof.

3. The method of claim 2, wherein the BoNT / A1 light chain or a variant thereof is a BoNT / A1 light chain variant, wherein the variant contains one or more amino acid substitutions, deletions or insertions in the membrane localization domain (MLD) (amino acid residues 275-334), and wherein the variant exhibits a BoNT activity duration altered compared to wild-type BoNT / A1.

4. The method of claim 3, wherein the BoNT / A1 light chain variant comprises a low homology domain of BoNT / A3, and / or comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

9.

5. The method of claim 1, wherein the BoNT light chain is a light chain of BoNT / A3 or a variant thereof.

6. The method of claim 5, wherein the BoNT / A3 light chain or a variant thereof is a BoNT / A3 light chain variant, wherein the variant contains one or more amino acid substitutions, deletions or insertions in the membrane localization domain (MLD), and wherein the variant exhibits an altered BoNT duration of action compared to wild-type BoNT / A3.

7. The method of claim 6, wherein the BoNT / A3 light chain variant comprises a low homology domain of BoNT / A3, and / or comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

11.

8. The method of claim 1, wherein the BoNT or SNARE cleaves the homologous light chain or a variant thereof as a light chain of BoNT / E or a variant thereof, or a light chain of BoNT / F or a variant thereof, or a light chain of BoNT / B or a variant thereof, or a light chain of BoNT / C or a variant thereof, or a light chain of BoNT / D or a variant thereof, or a light chain of BoNT / G or a variant thereof, or a light chain of BoNT / Wo or a variant thereof, or a light chain of BoNT / Ef or a variant thereof, or a light chain of BoNT / X or a variant thereof, or a light chain of TeNT or a variant thereof, or a light chain of any other member of the SNARE cleaving family of toxins.

9. The method of claim 8, wherein the BoNT light chain or a variant thereof is the BoNT / E light chain variant or the BoNT / F light chain variant.

10. The method of claim 9, wherein the BoNT / E light chain variant or the BoNT / F light chain variant contains one or more amino acid substitutions, deletions, or insertions in their respective membrane localization domains (MLDs).

11. The method of claim 10, wherein the BoNT / E light chain variant and / or the BoNT / F light chain variant comprises a low homology domain of BoNT / A1, and / or wherein the BoNT / E light chain variant and / or the BoNT / F light chain variant exhibit an increased duration of action compared to the wild-type BoNT / E light chain and / or the wild-type BoNT / F light chain.

12. The method of claims 1-11, wherein the synthesized mRNA construct is formulated into lipid nanoparticles.

13. The method of claim 12, wherein the lipid nanoparticles are tailored for targeting neuronal cells for therapeutic purposes.

14. A synthetic mRNA construct for producing clostridial neurotoxin (BoNT) or SNARE-cleaving homologous light chains or variants thereof.

15. The synthetic mRNA construct of claim 14, wherein the BoNT or SNARE cleavage homolog light chain or a variant thereof is a light chain of BoNT / A1 or a variant thereof.

16. The synthetic mRNA construct of claim 15, wherein the BoNT / A1 or SNARE cleavage homolog light chain or a variant thereof is a variant of the BoNT / A1 light chain, wherein the variant contains one or more amino acid substitutions, deletions or insertions in the membrane localization domain (MLD) (amino acid residues 375-334), and wherein the variant exhibits an altered duration of BoNT action compared to wild-type BoNT / A1.

17. The synthetic mRNA construct of claim 16, wherein the BoNT / A1 light chain variant comprises a low homology domain of BoNT / A3, and / or comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

9.

18. The synthetic mRNA construct of claim 14, wherein the BoNT or SNARE cleavage homolog light chain or a variant thereof is a light chain of BoNT / A3 or a variant thereof.

19. The synthetic mRNA construct of claim 18, wherein the BoNT or SNARE cleavage homologous light chain or a variant thereof is a BoNT / A3 light chain variant, wherein the variant contains one or more amino acid substitutions, deletions or insertions in the membrane localization domain (MLD), and wherein the variant exhibits an altered BoNT duration of action compared to wild-type BoNT / A3.

20. The synthetic mRNA construct of claim 19, wherein the BoNT / A3 light chain variant comprises a low homology domain of BoNT / A1, and / or comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

11.

21. The synthetic mRNA construct of claim 14, wherein the BoNT or SNARE cleaves a homologous light chain or a variant thereof as a light chain of BoNT / E or a variant thereof, or a light chain of BoNT / F or a variant thereof, or a light chain of BoNT / B or a variant thereof, or a light chain of BoNT / C or a variant thereof, or a light chain of BoNT / D or a variant thereof, or a light chain of BoNT / G or a variant thereof, or a light chain of BoNT / Wo or a variant thereof, or a light chain of BoNT / Ef or a variant thereof, or a light chain of BoNT / X or a variant thereof, or a light chain of TeNT or a variant thereof, or a light chain of any other member of the toxin family cleaved by SNARE.

22. The synthetic mRNA construct of claim 21, wherein the BoNT light chain or a variant thereof is the BoNT / E light chain variant or the BoNT / F light chain variant.

23. The synthetic mRNA construct of claim 22, wherein the BoNT / E light chain variant or the BoNT / F light chain variant contains one or more amino acid substitutions, deletions or insertions in their respective membrane localization domains (MLDs).

24. The synthetic mRNA construct of claim 23, wherein the BoNT / E light chain variant and / or the BoNT / F light chain variant comprises a low homology domain of BoNT / A1, and / or wherein the BoNT / E light chain variant and / or the BoNT / F light chain variant exhibit an increased duration of action compared to the wild-type BoNT / E light chain and / or the wild-type BoNT / F light chain.

25. A composition comprising a synthetic mRNA construct as described in any one of claims 14-24.

26. The composition of claim 25, wherein the synthesized mRNA construct is formulated into lipid nanoparticles or any suitable delivery carrier.

27. A BoNT / A3 variant comprising a heavy chain (HC) and a light chain (LC), wherein the LC comprises a low homology domain (LHD) of BoNT / A1.

28. The BoNT / A3 variant of claim 27, wherein the LC comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO:

11.

29. A BoNT / A1 variant comprising a heavy chain (HC) and a light chain (LC), wherein the LC comprises a low homology domain (LHD) of BoNT / A3.

30. The BoNT / A1 variant of claim 29, wherein the LC comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO:

9.

31. A method of treating a subject, the method comprising administering a therapeutically effective amount of a synthetic mRNA construct as described in any one of claims 14-24 or a composition as described in claims 25 and / or 26.

32. A synthetic mRNA construct for producing clostridial neurotoxin (BoNT) light chain (LC) or a variant thereof.

33. The synthetic mRNA construct of claim 32, wherein the BoNT LC or its variants include variants of BoNT / A1LC, BoNT / A3LC, BoNT / BLC, BoNT / CLC, BoNT / DLC, BoNT / ELC, BoNT / FLC, or BoNT / GLC.

34. The synthetic mRNA construct of claim 33, wherein the variants of BoNT / A1 LC, BoNT / A3 LC, BoNT / BLC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC or BoNT / G LC include domain substitutions.

35. The synthetic mRNA construct of claim 34, wherein the variant is a variant of BoNT / B LC, BoNT / CLC, BoNT / D LC, BoNT / E LC, BoNT / F LC or BoNT / G LC.

36. The synthetic mRNA construct of claim 35, wherein the domain substitutions of the BoNT / B LC, BoNT / C LC, BoNT / DLC, BoNT / E LC, BoNT / F LC or BoNT / G LC variants include a substituted N-terminus, a substituted membrane localization domain (MLD), or both.

37. The synthetic mRNA construct of claim 36, wherein the variants of BoNT / B LC, BoNT / C LC, BoNT / DLC, BoNT / E LC, BoNT / F LC or BoNT / G LC comprise a substituted N-terminus from BoNT / A1 or BoNT / A3, and a substituted MLD from BoNT / A1 or BoNT / A3.

38. The synthetic mRNA construct of claim 36, wherein the variant of BoNT / B LC, BoNT / C LC, BoNT / DLC, BoNT / E LC, BoNT / F LC or BoNT / G LC comprises a substituted N-terminus from BoNT / A1 or BoNT / A3.

39. The synthetic mRNA construct of claim 36, wherein the variants of BoNT / B LC, BoNT / C LC, BoNT / DLC, BoNT / E LC, BoNT / F LC or BoNT / G LC comprise a substituted MLD derived from BoNT / A1 or BoNT / A3.

40. The synthetic mRNA construct according to any one of claims 35-39, wherein the duration of action of the BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G LC is increased compared to wild-type BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G LC.

41. The synthetic mRNA construct of claim 34, wherein the variant is a variant of BoNT / A1 LC, and wherein the duration of action of the BoNT / A1 LC variant is shorter than the duration of action of wild-type BoNT / A1 LC.

42. The synthetic mRNA construct of claim 41, wherein the domain substitutions in the BoNT A1 LC variant include a substituted N-terminus from BoNT / A3, a substituted membrane localization domain (MLD) from BoNT / A3, or both.

43. The synthetic mRNA construct of claim 34, wherein the variant is a variant of BoNT / B LC, BoNT / D LC, BoNT / F LC or BoNT / G LC.

44. The synthetic mRNA construct of claim 43, wherein the variant of BoNT / B LC, BoNT / D LC, BoNT / F LC or BoNT / G LC comprises a substituted N-terminus and / or a substituted MLD from the N-terminus or MLD of one of the BoNT / B LC, BoNT / D LC, BoNT / F LC or BoNT / G LC, and wherein the variant of BoNT / B LC, BoNT / D LC, BoNT / F LC or BoNT / G LC exhibits altered potency and / or duration of action compared to the corresponding wild-type BoNT / B LC, BoNT / D LC, BoNT / F LC or BoNT / G LC.

45. A therapeutic composition comprising a clostridium neurotoxin (BoNT) variant, wherein the BoNT variant includes N-terminal, membrane localization domain (MLD), or both domain substitutions.

46. ​​The therapeutic composition of claim 45, wherein the BoNT variant includes variants of BoNT / A1, BoNT / A3, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G.

47. The therapeutic composition of claim 46, wherein the BoNT variant is a variant of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G.

48. The therapeutic composition of claim 47, wherein the variant of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F or BoNT / G comprises a substituted N-terminus from BoNT / A1 or BoNT / A3, and a substituted membrane localization domain (MLD) from BoNT / A1 or BoNT / A3.

49. The therapeutic composition of claim 47, wherein the variant of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F or BoNT / G comprises a substituted N-terminus from BoNT / A1 or BoNT / A3.

50. The therapeutic composition of claim 47, wherein the variants of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G comprise substituted MLDs derived from BoNT / A1 or BoNT / A3.

51. The therapeutic composition according to any one of claims 45-50, wherein the duration of action of the BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G variants is increased compared to the duration of action of wild-type BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G.

52. The therapeutic composition of claim 46, wherein the variant is a variant of BoNT / A1, and wherein the duration of action of the BoNT / A1 variant is shorter than the duration of action of wild-type BoNT / A1.

53. The therapeutic composition of claim 52, wherein the domain substitution in the BoNT A1 variant comprises a substituted N-terminus from BoNT / A3, a substituted membrane localization domain (MLD) from BoNT / A3, or both.

54. The therapeutic composition of claim 46, wherein the variant is a variant of BoNT / B, BoNT / D, BoNT / F or BoNT / G.

55. The therapeutic composition of claim 54, wherein the variant of BoNT / B, BoNT / D, BoNT / F, or BoNT / G comprises a substituted N-terminus and / or a substituted MLD from the N-terminus or MLD of one of BoNT / B, BoNT / D, BoNT / F, or BoNT / G, and wherein the variant of BoNT / B, BoNT / D, BoNT / F, or BoNT / G exhibits altered potency and / or duration of action compared to the corresponding wild-type BoNT / B, BoNT / D, BoNT / F, or BoNT / G.

56. A therapeutic composition comprising a synthetic mRNA construct as described in any one of claims 32-44.

57. The therapeutic composition of claim 56, wherein the synthesized mRNA construct is formulated into lipid nanoparticles or any suitable delivery carrier.

58. A method of treating a subject, the method comprising administering a therapeutically effective amount of a synthetic mRNA construct as described in any one of claims 32-44, a therapeutic composition as described in any one of claims 45-56, or a therapeutic composition as described in claim 56 or 57.

Images