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

Abstract

Disclosed herein are Clostridium neurotoxin (BoNT) or SNARE cleaving homologue variants, constructs, and methods of use. The BoNT or SNARE cleaving homologue variants can include one or more amino acid or domain substitutions, deletions, or insertions in the light chain.

Description

Atty. Dkt. No.650053.01105 PCT APPLICATION BOTULINUM NEUROTOXIN VARIANTS, CONSTRUCTS, COMPOSITIONS, AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 584,393 filed September 21, 2023, and U.S. Provisional Application No. 63 / 617,936 filed January 05, 2024, both of which are incorporated herein by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R01AI139306 by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (65005301014.xml; Size: 134,395 bytes; and Date of Creation: September 22, 2024) is herein incorporated by reference in its entirety. BACKGROUND

[0004] Clostridium botulinum are Gram-positive anaerobic bacteria that produce botulinum neurotoxins (BoNTs), the most toxic human proteins. BoNTs are 150 kDa zinc- dependent metalloprotease, possessing a 50-kDa N-terminal catalytic Light Chain (LC) and a 100-kDa C-terminal Heavy Chain (HC). The HC comprises a LC translocation domain (HCN) and a receptor binding domain (HCC), which enable neuronal cell association followed by endocytosis and translocation of the LC into the cell cytosol, respectively. BoNT includes seven pathogenic serotypes (A-G); BoNT / A includes eight subtypes (BoNT / A(1-8), which specifically cleave SNaptosomal-Associated Protein of 25 kDa (SNAP-25) on the cytoplasmic side of the neuronal plasma membrane. SUMMARY

[0005] In one aspect, a method is provided. The method can include administering a synthetic mRNA construct for producing a clostridial neurotoxin (BoNT) or SNARE cleaving homologue light chain or a variant thereof to a subject in need thereof.

[0006] In another aspect, a synthetic mRNA construct for producing a clostridial neurotoxin (BoNT) or SNARE cleaving homologue light chain or a variant thereof is provided.

[0007] In yet another aspect, a method of treating a subject is provided. The method can include administering a therapeutically effective amount of one or more of the synthetic mRNA constructs disclosed herein. QB\650053.01105\92299987.1 Page 1 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0008] In another aspect, a synthetic mRNA construct for producing a clostridial neurotoxin (BoNT) light chain (LC) or a variant thereof is provided.

[0009] In yet another aspect, a therapeutic composition is provided that includes one or more of the synthetic mRNA constructs disclosed herein.

[0010] In another aspect, a therapeutic composition is provided. The therapeutic composition can include a clostridial neurotoxin (BoNT) variant, where the BoNT variant comprises a domain substitution of an N-terminus, a membrane localization domain (MLD), or both.

[0011] In yet another aspect, a method for treating a subject is provided. The method can include administering a therapeutically effective amount of one or more the mRNA constructs disclosed herein and / or one or more of the therapeutic compositions disclosed herein. BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG. 1. Cytoskeleton inhibitors reveal LC / A1 traffics along microtubules. After overnight transfections, N2A cells were treated with a media control, 4µM of cytochalasin D or 9µM of Nocodazole 60 min prior to fixation with 4% paraformaldehyde. N2A cells were imaged for EGFP fluorescence (excitation 488 nm, emission 509 nm), phalloidin (excitation 555 nm, emission 605 nm), and ß-tubulin (excitation 645 nm, emission 705 nm). Representative images show the steady-state intracellular localization of EGFP, EGFP-LC / A1, EGFP-LC / A3V, and EGFP-LC / A3V.

[0013] FIG.2A and 2B. LC / A1 colocalizes with Rab GTPases. (FIG.2A) After overnight transfections and fixation with 4% paraformaldehyde and staining for Rab3a, / 4, / 5, / 7, / 11, and / 27a; N2A cells were imaged for EGFP fluorescence (excitation 488 nm, emission 509 nm), and α-Rab (excitation 645 nm, emission 705 nm). Representative images show steady-state intracellular localization and colocalization with the RAB GTPases indicated with white dots. (FIG. 2B) PCC of LC / A1 with the indicated Rab: 3a, 4, 5, 7, 11, or 27a and graphed with Graphpad Prism 9.3.1. Results were averaged from three independent experiments in triplicate.

[0014] FIG.3A and 3B. Dominant Negative Rab GTPases sequester LC / A1 to intracellular vesicles. (FIG. 3A) After overnight cotransfection of EGFP-Dominant-Negative Rab protein and DsRed-A1 were fixated with 4% paraformaldehyde; N2A cells were imaged for EGFP- DN Rab fluorescence (excitation 488 nm, emission 509 nm), and DsRed-A1 fluorescent (excitation 555 nm, emission 605 nm). Representative images show the steady-state intracellular localization of EGFP-Dominant-Negative Rab protein and DsRED-A1 individually and merged. Control of EGFP-LC / A1 and DsRed-LC / A1 was analyzed to ensure QB\650053.01105\92299987.1 Page 2 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION DsRed-LC / A1 did not alter LC / A1's membrane localization. (FIG. 3B) Percentage of cells containing membrane signal. Ten random fields were selected and counted for membrane localization from three independent experiments. Mean, and SEM was evaluated with GraphPad Prism 9.3.1 using ordinary one-way ANOVA with Dunnett's multiple comparisons test with DsRed-A1 as the control column. ns- not significant; all other values are **** p<0.0001. Results were averaged from three independent experiments in triplicate.

[0015] FIG. 4. Trafficking of Botulinum neurotoxin LC / A1 after translocation in Neuro- 2A cells. (1) BoNT / A1 binds a ganglioside and a synaptic vesicle protein-2 (SV2) on the surface of a motor neuron. (2) BoNT / A is endocytosed in a Rab 5 early endosome (3) then trafficked to a sorting endosome where (4a) BoNT / A1 is trafficked into Rab4 fast recycling endosomes, upon acidification, LC / A1 translocates through the pore created by the HCC. (4b) BoNT / A1 is trafficked into Rab11 recycling endosomes, which facilitate slow recycling as DN Rab11 does not inhibit LC / A1 membrane localization; this pathway is secondary to Rab4 recycling. (4c) A portion of BoNT / A1 is trafficked into Rab7 late endosomes and retrograde trafficked towards the nucleus. (5) After translocation, LC / A1 facilitated by the N terminus (N) (red, residues 1-17) associates with Rab3 and Rab 27 SVs for (6) anterograde trafficking along microtubules associated with the plasma membrane (7) where the low homology domain (LHD) (green, residues 275-357 A1) transitions LC / A1 by recognizing SNAP-25 for localization and cleavage. 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, recycling endosome; SE, sorting endosome; SV2, Synaptic Vesicle Protein-2.

[0016] FIG. 5. Determining boundaries for Experimental Pearson's colocalization coefficients. (Left) N2A cells were transfected with EGFP-LC / A1, then incubated α-EGFP antibody and stained for DNA utilizing DAPI. Representative deconvolved epifluorescence images are shown. Scale bar, 5 µm. (Right) Pearson's colocalization coefficients are graphed using GraphPad Prism 9.3.1.

[0017] FIG. 6. EGFP-TRAP Coimmunoprecipitation of EGFP-LC constructs. The protocol was utilized as described. Cell lysates of N2A cells transfected the DNA encoding the indicated EGFP-fusion protein (*) was subjected to EGFP-TRAP coimmunoprecipitation, samples were run on a 13.5% SDS-PAGE, transferred to a PVDF membrane, and probed for EGFP, actin, and ß-3-tubulin, with1:2000 rat α-EGFP-monoclonal IgG (1:20000 mouse α-ß- actin monoclonal IgG, or 1:2000 rabbit α-ß-3 tubulin. Bound primary antibodies were recognized with Goat-α-rat 1:10,000, Goat-α-mouse 1:20000, or Goat-α-rabbit 1:10,000, conjugated with horseradish peroxidase with Super Signal for detection as described in the QB\650053.01105\92299987.1 Page 3 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION methods section. The EGFP signal was detected in lysates expressing EGFP-fusion protein ~75 kDa, while lysates expressing EGFP alone yielded an (EGFP) reactive band at ~26 kDa, actin reactive band ~42 kDa, and ß-3 tubulin reactive band ~50 kDa.

[0018] FIG. 7. Dominant Negative Rab GTPases affect Ctx-B and Transferrin internalization. (Left) After overnight cotransfection of EGFP-Dominant-Negative Rab, N2A cells were treated with cholera toxin b subunit (Ctx-B) for 5 min at 37 °C; cells were then fixated with 4% paraformaldehyde; N2A cells were imaged for EGFP fluorescence (excitation 488 nm, emission 509 nm), and Ctx-B (excitation 645 nm, emission 702 nm). Representative images show the steady-state intracellular localization of EGFP-Dominant-Negative Rab GTPases and Ctx-B individually and merged. (Right) After overnight cotransfection of EGFP- Dominant-Negative Rab GTPases were treated with transferrin for 5 min at 37 °C; cells were then fixated with 4% paraformaldehyde; N2A cells were imaged for EGFP fluorescence (excitation 488 nm, emission 509 nm), and transferrin (excitation 645 nm, emission 705 nm). Representative images show the steady-state intracellular localization of EGFP-Dominant- Negative Rab GTPases and transferrin individually and merged.

[0019] FIG. 8. Coomassie stained gel of purified rA3 Q7P, V14G toxin: Purified recombinant BoNT / A3- Q7P, V14G was either reduced with DTT (R) or not reduced (NR) and separated by SDS-PAGE gels and stained with InstaBlue (MidSci) total protein stain. A protein ladder was run alongside the protein (Seeblue2, Life Technologies) for molecular weight determination of the protein bands. The BoNT / A3- Q7P, V14G was almost fully reduced to the 100 and 50 kDa heavy chain and light chain bands, indicating proper di-chain formation.

[0020] FIG.9. In vivo duration of action of BoNT / A3 and BoNT / A3 - Q7P, V14G. Mice were injected into the gastrocnemius muscle with the indicated doses of either BoNT / A3 and BoNT / A3 - Q7P, V14G, and overall motor-neuron deficiency was monitored by Rotarod and local paralysis was monitored by DAS assay. N=5.

[0021] FIG. 10. Duration of action of BoNT / A3 and BoNT / A3- Q7P, V14G in primary neurons. Primary rat spinal cord cells were exposed to either BoNT / A3 wt or BoNT / A3- Q7P, V14G, and samples of the cells were harvested at the indicated time points in triplicate. Cell lysates were analyzed by Western blot and densitometry for the percentage of SNAP-25 that had been cleaved by BoNT. The percent un-cleaved SNAP-25 is shown over time, reflecting recovery of the cells from the BoNT intoxication.

[0022] FIG.11. BoNT LC activity in motor-neurons transfected with mRNAs. The hiPSC derived motor-neurons were transfected with the indicated mRNA constructs, and samples of QB\650053.01105\92299987.1 Page 4 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION cells (n=5) were harvested at the indicated time points. The average and standard deviations of the percent cleaved SNAP-25 at each time point was determined, and the data points were fitted with logarithmic trend-lines.

[0023] FIG. 12. Transfection of human motor-neurons with LNP formulated LC / A1 mRNA. The hiPSC derived motor-neurons were exposed to the ciLNP formulated LC / A1 mRNA for the indicated amounts of time, and cell lysates were analyzed for SNAP-25 cleavage by Western blot and densitometry (n=2).

[0024] FIGS. 13A-13C. FIG. 13A depicts the three dimensional structure of BoNT / A1 light chain, FIG, 13B depicts a close up view of a portion of the MLD domain. FIG.13C is a sequence alignment of the MLD regions of various BoNT serotypes. In descending order Fig. 13C sequences are SEQ ID NO: 56-66.

[0025] FIG. 14. BoNT LC activity in motor-neurons transfected with mRNAs in LNPs. The hiPSC derived motor-neurons were transfected with the indicated mRNA constructs, and samples of cells (n=5) were harvested at the indicated time points. The average and standard deviations of the percent cleaved SNAP-25 at each time point was determined, and the data points were fitted with logarithmic trend-lines.

[0026] FIG. 15A-15C. LC / A1 traffics along microtubules colocalized with Rab GTPase. (A) After overnight transfection with pEGFP-LC / A1, pEGFP-LC / A3, pEGFP, or untreated (Control), N2A cells were treated with cytochalasin D (Cytochalasin D), Nocodazole (Nocodazole), or not treated (Control), 60 min prior to fixation with 4% paraformaldehyde. N2A cells were imaged for EGFP fluorescence (green; excitation 488 nm, emission 509 nm), phalloidin (actin, phalloidin-Alexa568, red; excitation 555 nm, emission 605 nm), and ß- tubulin (microtubules, primary: rabbit α-ß tubulin followed with goat-anti-rabbit-Alexa647 magenta; excitation 645 nm, emission 705 nm). Representative images show the steady-state 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 nocodazole. Starting at seven hours post pEFGP-LC / A1 transfection, N2A cells were treated with Cytochalasin D for 60 min, then live-imaged every 15 sec for 15 min. EGFP-LC / A1 bi- directional movement in neurite sprouts was detected, data not shown. (B) After overnight transfections with pEGFP-LC / A1, N2A cells were fixed with 4% paraformaldehyde then probed for individual Rab GTPases (primary: rabbit α-Rab IgG, followed with secondary goat- anti-rabbit-Alexa568) and imaged for EGFP-LC / A1 (EGFP: green) and individual Rab3a, Rab4, Rab5, Rab7, Rab11, or Rab27a α-Rab: red). Representative images show steady-state intracellular localization of individual Rab GTPases (Rab), EGFPLC / A1 (GFP-LC / A1), merge QB\650053.01105\92299987.1 Page 5 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION of individual Rab GTPases and EGFP-LC / A1 (Merge), and colocalization of individual Rab GTPases and EGFP-LC / A1 indicated with white dots (Colocalized). FIG. 15C Pearson’s Colocalization Coefficient (PCC) of LC / A1 with the indicated Rab3a, Rab4, Rab5, Rab7, Rab11, or Rab27a. Controls to establish colocalization (anti-GFP) and segregation (DNA) with EGFP-LC / A1 are indicated by dashed columns and were analyzed in a separate experiment (FIG. 17). The data were graphed with GraphPad Prism 10.1.2. Results were averaged from three independent experiments in triplicate.

[0027] FIG.16A-16B. Dominant Negative Rabs involved in SV cycling inhibit N-terminal dependent LC / A1 trafficking to the plasma membrane. After overnight co-transfections of pEGFP-Dominant-Negative Rabs and pDsRed-LC / A1 FIG. (A) or pDsRed-LC / A3 (A1-MLD) (B), N2A cells were fixated with 4% paraformaldehyde and imaged for EGFP-DN Rab fluorescence (green), and DsRed-LC / A fluorescence (red). Representative images show steady-state intracellular localization of individual EGFP-DN Rabs (GFP-DN Rab) and DsRed- LC / A1 (A) or pDsRed-LC / A3 (A1-MLD) (B) and merged (Merged). Percentage of cells containing membrane localized DsRed-LC / A1 fluorescence alone or DsRed-LC / A3 (A1- MLD) alone or in the presence of individual DN Rab were analyzed from ten random fields in three independent experiments. Typical observed plasma membrane localization of DsRed- LC / A3 (A1-MLD) in the presence of the individual DN Rab GTPases is marked with a red arrow). Mean and SEM were evaluated with GraphPad Prism 10.1.2 and subjected to statistical analysis with an ordinary one-way ANOVA with Dunnett’s multiple comparisons test using % membrane localized DsRed-LC / A1 (alone) or DsRed-LC / A3V (A1 MLD) (alone) as the control column. ns = not significant, *** p<0.001.

[0028] Figure 17A-17C. Establishing colocalization and segregation values for Pearson’s colocalization coefficients (PCC). (A) After overnight transfections with pEGFP-LC / A1, N2A cells were fixed with 4% paraformaldehyde then probed for EGFP (primary: rat α-GFP IgG followed by goat-anti-rat IgG-Alexa647 (purple) and stained for DNA (Hoechst, blue excitation 405 nm and emission 455 nm). Representative fluorescent images are shown. (B) Representative images for EGFP-LC / A1 with α-EGFP (colocalization) or DNA (segregated) were analyzed with Image J for colocalization, scatterplots and Pearson's colocalization coefficients (PCC), indicate segregated pixels (purple / green or green / blue) and the overlapping pixels (white) between the two channels, were graphed with GraphPad Prism 10.1.2, and PCC were graphed with GraphPad Prism 10.1.2. (C) The average PCC of at least 5 control cells were analyzed and graphed using GraphPad Prism 10.1.2. QB\650053.01105\92299987.1 Page 6 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0029] FIG.18A-18B. Dominant Negative Rabs Sequester CT / B and Transferrin to unique intracellular locations. After overnight co-transfections of individual pEGFP-Dominant- Negative Rabs, N2A cells were treated with (A) DsRed-cholera toxin b subunit (CT / B) or (B) transferrin for 5 min at 37 °C; then fixed with 4% paraformaldehyde and imaged for EGFP fluorescence (excitation 488 nm, emission 509 nm), and CT / B (Cholera Toxin B subunit- Alexa-Fluor 594 nm (red) or transferrin (transferrin-Alexa-Fluor 594 (red). Representative images show steady-state intracellular localization of EGFP-DN Rab GTPases (DN Rab) and CT / B-Alexa-Fluor 594 (CT / B) or Transferrin-Alexa-Fluor 594 (Transferrin) individually and merged (Merge).

[0030] FIG.19A-19G. Dominant Negative Rabs involved in SV cycling inhibit N-terminal mediated LC / A1 cleavage of plasma membrane bound SNAP-25. After an overnight transfection with the indicated, individual pEGFP-DN Rabs, pEGFP, and (A-B) pEGFP- LC / A1 or (C) pEGFP-LC / A3V(A1-MLD), N2A cells lysates were prepared and subjected to SDS-PAGE, then transferred to PVDF membranes, and probed with rat α-EGFP monoclonal IgG (primary rat α-GFP) (A, C) and mouse-α-SNAP-25 monoclonal IgG (primary: rat α- SNAP-25) (A). Bound primary antibodies were recognized with goat-α-rat-HRP (A, C) or goat- α-mouse-HRP (A) respectively. Bound HRP-conjugated secondary antibodies were detected with Super Signal. EGFP-LC / A1 was detected as a 75-kDa protein (A1) (A, C), individual DN Rabs as a 50-kDa (C), EGFP as a 25-kDa (C), or uncleaved (U)- and cleaved (C)- SNAP-25 were detected as a~25-kDa proteins (A). (B) Data from (A) was quantified by densitometry and graphed using GraphPad 10.1.2. After an overnight co-transfection with plasmids encoding individual EGFPDN Rab GTPases with (D, E) EGFP-LC / A1 or (F, G) EGFP-LC / A3V (A1 MLD) (F, G), and carrier EGFP, N2A cells were fixed with 4% paraformaldehyde and stained for cleaved SNAP-25 (residues 1-197) with mouse-anti-SNAP-25 (1-197) IgG (red, primary) followed by goat-α-mouse IgG-Alexa568). N2A cells were imaged for EGFP expression (GFP fluorescence (107) arbitrary units (AU), green), and cleaved SNAP-25 (cleaved SNAP-25 fluorescence (107) AU, red). Representative N2A cells transfected with (D) EGFP-LC / A1 or (F) EGFP-LC / A3V (A1 MLD). The sum fluorescence intensity for individual cells in panel E was ~3X107AU (GFP), and (F) was~1.5X107AU (GFP), respectively, were scored for cleaved SNAP-25 (red) and merged (EGFP (green)). LUTS for the immunofluorescent panels were set for EGFP-A1 (D) (453 min & 3251 max), EGFP-A3V (A1 MLD) (F) (299 min & 1247 max), and cleaved SNAP-25 (736 min and 1344 max) (D) (433 min and 958 max) (F), merged lane contained LUTs based on EGFP fluorescence and cleaved SNAP-25. (E, G) Individual cells were analyzed from three independent experiments for EGFP X 107AU (X-axis) and cleaved QB\650053.01105\92299987.1 Page 7 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION SNAP-25 X 107AU (Y-axis) sum intensity fluorescence and plotted in GraphPad Prism 10.1.2. Slopes of linear regression lines for EGFP-LC / A1 (E) or EGFP-LC / A3V (A1 MLD) (G): EGFP alone (dotted black), and EGFP + EGFP-LC / A1 (purple), EGFP + EGFP-LC / A3V (A1 MLD) (teal), and EGFP + EGFP-LC / A1 + the indicated DN Rab (orange). Statistical analysis was performed on the slope of EGFP + EGFP-LC / A1 vs the slope of EGFP + EGFP-LC / A1 + the indicated DN Rab GTPase (E) or of EGFP + EGFP-LC / A3V (A1 MLD) vs the slope of EGFP +EGFP-LC / A3V (A1 MLD) + the indicated DN Rab GTPase (G). ns = not significant; *** p<0.001, or **** p<0.0001.

[0031] FIG 20A-20H. Intracellular localization via the N terminus and MLD contribute to LC / A potency and stability as the basis for duration of action. (A-B) Human motor neurons were transfected overnight with two-fold serial dilutions with mRNA encoding LC / A1, LC / A3, or LC / A3V. After 48 h, cells were washed and in cubations continued for the indicated times when lysates were prepared, subjected to SDS-PAGE, transferred to PVDF membranes, and probed with mouse-α-SNAP-25 monoclonal IgG (primary: rat α-SNAP-25) which detects LC / A cleaved and uncleaved SNAP-25, bound antibody was detected with goat-α-mouse-HRP (A) respectively and visualized with Super Signal. Densitometry from at least three independent experiments on cleaved and uncleaved SNAP-25 were plotted as the % of uncleaved / cleaved SNAP-25 was plotted vs the time of incubation. (C-E) N2A cells were transfected overnight with two-fold serial dilutions of DNA encoding EGFP-LC / A1 (125-3.9 ng), EGFP-LC / A3 (125-3.9 ng) or pEGFP-LC / A3V (2000-62.5 ng). Cells lysates were prepared and subjected to SDS-PAGE, transferred to PVDF membranes, probed with rat α- EGFP monoclonal IgG (primary rat α-GFP) and mouse-α-SNAP-25 monoclonal IgG (primary: rat α-SNAP-25). Bound primary antibodies were recognized with goat-α-rat-HRP (A, C) or goat-α-mouse-HRP (A) respectively and detected with Super Signal. Densitometry from at least three independent experiments on cleaved and uncleaved SNAP-25 were normalized to overall LC / A expression and graphed utilizing GraphPad Prism 10.1.2. (B table-C graph) Specific activity of LC / A1, / A3, and / A3V at 30% cleaved SNAP-25 averaged from at least three independent experiments graphed utilizing GraphPad Prism 10.1.2 and ns = not significant, *** p<0.001, *** p<0.001. Statistical analysis from average of at least three independent experiments normalized to LC / A specific 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). Within experiment (A), N2A cells were also transfected overnight with two-fold serial dilutions of DNA encoding pEGFP-LC / A3V(A1-N1), pEGFP-LC / A3V(A1-MLD), or pEGFP- QB\650053.01105\92299987.1 Page 8 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION LC / A3V(A1-N1,A1-MLD) (125-3.9 ng). Cells lysates were prepared and probed for EGFP and cleaved and uncleaved SNAP-25 were normalized to overall LC / A expression and graphed utilizing GraphPad Prism 10.1.2 as described in (A), and outcomes were plotted over the data in (A). In vivo duration of action of BoNT / A3 and BoNT / A3(V). Mice were injected in the gastrocnemius muscle with the indicated doses of either BoNT / A3 and BoNT / A3(V), and overall motor-neuron deficiency was monitored by (G) Rotarod (Rotarod) and local paralysis was monitored by (H) DAS assay (DAS) N=5.

[0032] FIG. 21. Intracellular trafficking of LC / A1 to the plasma membrane to cleave SNAP-25 in Neuro-2A cells. Following LC / A1 synthesis in the cytosol of N2A cells, sensitivity of LC / A1 trafficking to the plasma membrane and SNAP-25 cleavage by DN Rab GTPases / 4, / 3a, / 27 and / 5 implicate productive LC / A1 intracellular trafficking via a fast Rab4- positive recycling endosomes to Rab3a / Rab27- synaptic vesicles, where LC / A1 is delivered to the plasma membrane. Rab5-positive early endosomes may function by production of upstream recycling endosomes that initiate Rab4-mediated vesicle trafficking (Solid Arrows). The model allows LC / A1 to enter the pathway at each-vesicle implicated in fast, SV recycling and that the pathway is sequentially linked for productive LC / A1 movement to the plasma membrane. Rab7-positive late endosomes (LE) and Rab11-positive slow RE which are downstream of Rab-5 mediated recycling endosomes do not contribute to LC / A1 trafficking to the plasma membrane or SNAP-25 cleavage (broken lines). Created with BioRender.com. DETAILED DESCRIPTION

[0033] Overview

[0034] Disclosed herein are clostridium neurotoxin (BoNT), tetanus toxin, or SNARE cleaving homologue variants, constructs, compositions, and methods of use.

[0035] In various aspects, the BoNT, tetanus toxin, or SNARE cleaving homologue or variant and constructs disclosed herein are designed to modulate the duration of BoNT or SNARE cleaving homologue action within a target cell. Duration of action is an important feature of BoNTs as a human therapeutic and as a component of botulism. The disclosure herein includes BoNT variants and constructs encoding such variants, that can include amino acid substitutions, insertions, or deletions, and / or partial or whole domain substitutions, deletions, or insertions to modulate the duration of BoNT action and / or BoNT potency. For instance, in certain aspects, the BoNT variants can include mutations and / or partial or whole domain substitutions with respect to the N-terminal domain and / or the membrane localization domain (MLD) present in the light chain of the BoNT protein, as described herein. For the sake of clarity, throughout this disclosure BoNT may be referred to in isolation but it should QB\650053.01105\92299987.1 Page 9 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION be understood that when discussing BoNT, other SNARE cleaving homologues are also contemplated, e.g., tetanus toxin.

[0036] In various aspects, the constructs disclosed herein provide for delivery of BoNT light chains (LC) or variants thereof to a subject. In one or more aspects, the constructs can be synthetic mRNA constructs encoding for a BoNT LC or variant thereof. In various aspects, the BoNT LC variants can include amino acid substitutions or partial or whole domain substitutions to modulate the duration of BoNT action. mRNA transfection for the delivery of genetically modified BoNT-LCs that have specific duration of actions are an alternative therapeutic that offers several advantages relative to the currently used injection of the BoNT / A1 protein. For instance, mRNA transfection is easier, safer, and less expensive to produce. Further, mRNA transfection for BoNT therapy involves less risk of developing immune resistance compared to administration of BoNT / A1. mRNA transfection for BoNT therapy can be used in patients that have developed immunity to BoNTs due to vaccination or therapeutic injections. In addition, mRNA transfection of BoNT LCs or variants thereof can allow for control of the target by injection site and route and the engineered mRNA complex, not by the binding of the toxin to neuronal cell surface proteins as is the case with current BoNT therapies. Also, mRNA constructs and methods disclosed herein allow for targeting of non- neuronal cells by the BoNT LCs modified to cleave non-neuronal SNARE targets. Yet further, the mRNA constructs disclosed herein allow for delivery of BoNT-LC into target cells as an exocytosis inhibitor, whereby the duration of action can be adjusted based on the mRNA sequence. In various aspects, any clostridial neurotoxin (or other SNARE cleaving homolog) light chain mRNA or truncated or extended version thereof can be used for therapeutic purposes, and are contemplated herein. Additional mRNA production or delivery methodology can be and is expected to be applied to deliver the mRNA to target cells in vivo, and the therapeutic use of the mRNA encoding a SNARE cleaving enzyme to treat diseases related to exocytosis is considered to be within the scope of the present disclosure, regardless of the delivery vehicle or mRNA design.

[0037] Definitions and Terminology

[0038] The disclosed botulinum neurotoxin (BoNT) variants, constructs, compositions, and methods, may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular aspects only and are not intended to be limiting.

[0039] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. QB\650053.01105\92299987.1 Page 10 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0040] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0041] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0042] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0043] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0044] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth. QB\650053.01105\92299987.1 Page 11 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0045] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0046] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g., of an entire nucleic acid or polypeptide sequence or individual portions or domains of a nucleic acid or polypeptide), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0047] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0048] An example of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. As will be appreciated by one of skill in the art, the software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information (NCBI). In embodiments, BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins. In embodiments, a BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the QB\650053.01105\92299987.1 Page 12 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION same length in a database sequence. In embodiments, T is referred to as the neighborhood word score threshold (Altschul et al., supra). In embodiments, these initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. In embodiments, the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. In embodiments, cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). In embodiments, for amino acid sequences, a scoring matrix is used to calculate the cumulative score. In embodiments, extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. In embodiments, the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. In embodiments, the NCBI BLASTN or BLASTP program is used to align sequences. In embodiments, the BLASTN or BLASTP program uses the defaults used by the NCBI. In embodiments, the BLASTN program (for nucleotide sequences) uses as defaults: a word size (W) of 28; an expectation threshold (E) of 10; max matches in a query range set to 0; match / mismatch scores of 1, −2; linear gap costs; the filter for low complexity regions used; and mask for lookup table only used. In embodiments, the BLASTP program (for amino acid sequences) uses as defaults: a word size (W) of 3; an expectation threshold (E) of 10; max matches in a query range set to 0; the BLOSUM62 matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)); gap costs of existence: 11 and extension: 1; and conditional compositional score matrix adjustment.

[0049] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0050] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is QB\650053.01105\92299987.1 Page 13 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0051] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0052] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0053] As to amino acid sequences, one of skill will recognize that individual substitutions to a peptide, polypeptide, or protein sequence which alters a single amino acid is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. QB\650053.01105\92299987.1 Page 14 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0054] The following eight groups each contain amino acids that are conservative substitutions for one another: 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, e.g., Creighton, Proteins (1984)).

[0055] The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human subjects. In some embodiments, subjects may be any mammal.

[0056] As used herein, the term “treating” refers to reducing, eliminating, improving the condition of, or lessening the severity of any aspect of a symptom benefiting from BoNT therapy in a patient. In various aspects the present disclosure includes a method of treating a patient having a symptom benefitting from BoNT therapy. In such cases, the method can comprise treating the patient with an effective amount of the BoNT variants and / or mRNA constructs provided herein whereby the symptom is reduced, eliminated or lessened.

[0057] As used herein, “therapeutically effective amount” refers to an amount of a therapeutic agent sufficient to effect beneficial or desirable biological and / or clinical results. The BoNT variants and mRNA constructs can be administered in a therapeutically effective amount depending upon the type of treatment necessary. Methods of determining suitable dosage or dosage ranges for individual treatment are known to those in the art. In various aspects, the BoNT variants and / or mRNA constructs can be administered as a single dose or, where appropriate, multiple doses. BoNT and other SNARE Cleaving Homologues, Variants, Constructs, and Compositions

[0058] In various aspects, BoNT variants are disclosed. In certain aspects, the BoNT variants can be variants from any BoNT serotype and / or subtype. As discussed above, in certain aspects, the BoNT variants disclosed herein can include one or more amino acid substitutions, deletions, insertions, or domain substitutions that can modulate the potency and / or duration of BoNT action. In certain aspects, the BoNT variants can include one or more mutations and / or whole or partial domain substitutions with respect to the N-terminus, the membrane localization domain (MLD), and / or the low homology domain (LHD) present in the light chain of the BoNT protein. One example MLD is the MLD of the BoNT / A1 LC found at amino acid residues 275-334 with reference to the BoNT / A1 LC sequence of 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 QB\650053.01105\92299987.1 Page 15 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION protein and / or variants of the light chain (LC) or the heavy chain (HC). In various aspects, other SNARE cleaving homologues and variants are disclosed, including but not limited to tetanus toxin. Table 1: N-terminal Domain, MLD, and LHD of various BoNTs BoNT:  N‐ MLD‐R1  MLD‐R2  MLD‐R1‐R2  LHD‐R1‐ LC amino  Holotoxin  Crystal  terminus  amino  amino  amino acid  R2‐R3  acid  amino  Structure  i id id id i id id id f ce     d  e.

[0059] As discussed herein, the BoNT variants and BoNT LC variants can include one or more substituted domains, in various aspects. A non-limiting list of potential domains for substitution in BoNTs is the N-terminus, the MLD, and the LHD. As used herein, a substituted domain refers to a reference BoNT having its wild type domain, e.g., MLD domain, removed and replaced with a MLD from another BoNT. As one example using Table 1 above, BoNT / A1 amino acid residues 275-334 (MLD) could be removed and replaced with amino acid residues 277-334 (MLD) of BoNT / A3, thereby providing a BoNT / A1 variant that includes a substituted MLD from BoNT / A3.

[0060] In aspects, the duration of BoNT action can be determined in various manners, e.g., in vivo in a mouse model and / or in vitro by assaying cultured cells. Both assays are described with reference to Examples 2 and 3 below, with results shown in FIGS. 9, 10, and 11. For instance, in in vivo mouse models one can assess BoNT duration of action utilizing the Rotarod assay and / or the digital abduction score assay. In vitro determination of BoNT duration of activity can be assessed by assaying for uncleaved and / or cleaved SNARE proteins. In various aspects, one can assay for cleaved or uncleaved SNARE proteins, such as, for example, VAMP, QB\650053.01105\92299987.1 Page 16 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION SNAP-25, and Syntaxin. In one aspect, in vitro determination of BoNT duration of activity can be assessed by assaying for uncleaved and / or cleaved SNAP -25 in lysed neurons.

[0061] In one example aspect, the BoNT / A3 protein can include the N-terminus, the MLD, and / or the LHD from the BoNT / A1 protein, which can decrease the duration of BoNT action. In certain aspects, the BoNT / A3 variant can 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: 11. In various aspects, the BoNT / A3 variant can comprise or consist of a light chain having an amino acid sequence that is the amino acid sequence of SEQ ID NO: 11.

[0062] In various aspects, the BoNT / A1 protein (or tetanus toxin (TT) protein, or homologues of BoNT or TT or other SNARE cleaving homologue) can include the N-terminus, the MLD, and / or the LHD of the BoNT / A3 protein, which can increase the duration of BoNT action. In certain aspects, the BoNT / A1 variant can 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 can comprise or consist of a light chain having an amino acid sequence that is the amino acid sequence of SEQ ID NO: 9.

[0063] In various aspects, any BoNT protein can be modified to include the N-terminus, the MLD, and / or the LHD from BoNT / A1, which may aid in increasing the duration of BoNT action. For instance, in one aspect, the BoNT / E protein can have its N-terminal domain, the MLD, and / or the LHD substituted for the N-terminus, the MLD, and / or the LHD from BoNT / A1, which allows for an increased duration of BoNT action. In certain aspects, the BoNT / F protein can have its N-terminus, the MLD, and / or the LHD substituted for the N- terminal domain, the MLD, and / or the LHD from BoNT / A1, which allows for an increased duration of BoNT action. The wild type BoNT / E and the BoNT / F proteins can enter cells faster than BoNT / A1, but the wild type BoNT / E and BoNT / F exhibit a short duration of BoNT action. Thus, by modifying the BoNT / E and / or the BoNT / F proteins with the N-terminus, the MLD, and / or the LHD of BoNT / A1, BoNT variants are produced that have fast cell entry and increased duration of action. It should be understood that other BoNT serotypes can also be modified to include the N-terminus, the MLD, and / or the LHD of the BoNT / A1 protein to modulate and / or increase the duration of BoNT action, such as, the BoNT / A2, A4, A5, A6 proteins, or the B, C, D, and G serotypes. Alternatively, any BoNT protein can include any amino acid substitution, deletion, or insertion to its N-terminus, the MLD, and / or the LHD to modulate the duration of BoNT action. QB\650053.01105\92299987.1 Page 17 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0064] In various aspects, the BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, and / or BoNT / G proteins or holotoxins can include a substitution of their N-terminus, the MLD, and / or the LHD with the respective N-terminus, the MLD, and / or the LHD from BoNT / A1 or BoNT / A3, to modulate the potency and / or duration of action.

[0065] In various aspects, the BoNT / B, BoNT / D, BoNT / F, and / or BoNT / G proteins or holotoxins can include a substitution of their N-terminus, the MLD, and / or the LHD with the respective N-terminus, MLD, and / or LHD of one or more of the remainder of the BoNT / B, BoNT / D, BoNT / F, and / or BoNT / G proteins or holotoxins. For instance, the BoNT / B holotoxin can include a domain substitution of the N-terminus, the MLD, and / or the LHD with the N- terminus, MLD, and / or LHD from BoNT / D, BoNT / F, or BoNT / G.

[0066] In various aspects, as discussed above, BoNT light chain (LC) variants are contemplated herein. For instance, in certain aspects, a BoNT / A3 light chain (LC) variant in the absence of the heavy chain is also contemplated, that can include the N-terminus, MLD, and / or LHD from BoNT / A1. In certain aspects, the BoNT / A3 LC variant can 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: 11. In various aspects, the BoNT / A3 LC variant can comprise or consist of a light chain having an amino acid sequence that is the amino acid sequence of SEQ ID NO: 11.

[0067] In certain aspects, the BoNT / B light chain (LC), BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, and / or BoNT / G LC can include a substitution of their N-terminus, the MLD, and / or the LHD with the respective N-terminus, the MLD, and / or the LHD from BoNT / A1 or BoNT / A3.

[0068] In various aspects, the BoNT / B LC, BoNT / D LC, BoNT / F LC, and / or BoNT / G LC can include a substitution of their N-terminus, the MLD, and / or the LHD with the respective N-terminus, MLD, and / or LHD of one or more of the remainder of BoNT / B, BoNT / D, BoNT / F, and / or BoNT / G. For instance, the BoNT / B LC can include a domain substitution of the N-terminus, the MLD, and / or the LHD with the N-terminus, MLD, and / or LHD from BoNT / D, BoNT / F, or BoNT / G.

[0069] In various aspects, the BoNT or SNARE cleaving homologue light chain or variant thereof may be light chain of BoNT / E or a variant thereof, or the light chain of BoNT / F or a variant thereof, or the light chain of BoNT / B or a variant thereof, or the light chain of BoNT / C or a variant thereof, or the light chain of BoNT / D or a variant thereof, or the light chain of BoNT / G or a variant thereof, or the light chain of BoNT / Wo or a variant thereof, or the light chain of BoNT / Ef or a variant thereof, or the light chain of BoNT / X or a variant thereof, or the QB\650053.01105\92299987.1 Page 18 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION light chain of TeNT or a variant thereof, or the light chain of any other member of the larger family of SNARE cleaving toxins.

[0070] In one or more aspects, a BoNT / A3 LC variant is disclosed that includes 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 mentioned herein for BoNT / A3 LC can also be present in full length BoNT / A3.

[0071] In various aspects, a BoNT / A1 light chain (LC) variant in the absence of the heavy chain is also contemplated, that can include the MLD and / or the LHD from BoNT / A3. In certain aspects, the BoNT / A1 LC variant can 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 LC variant can comprise or consist of a light chain having an amino acid sequence that is the amino acid sequence of SEQ ID NO: 9.

[0072] The BoNT variants, including the BoNT LC variants may be produced in various ways. For instance, the BoNT variants and / or the BoNT LC variants can be produced recombinantly in E. coli, yeast, or other convenient expression system.

[0073] As discussed above, in various aspects, synthetic mRNA constructs are disclosed herein. In various aspects, the synthetic mRNA constructs are for producing LCs of clostridial neurotoxins, e.g., BoNT, TT, or homologues thereof. In various aspects, the synthetic mRNA constructs are for producing a BoNT LC or LC variant thereof. In certain aspects, the mRNA constructs can encode for any of the BoNT LCs or LC variants disclosed herein. In various aspects, the mRNA constructs can encode for any BoNT LC, TT, or TT LC, or homologues of BoNT or TT.

[0074] In various aspects, the mRNA constructs can encode for 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 the amino acid sequence of 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 constructs can encode for any BoNT LC variant, or TT variant, or homologue of TT or BoNT, e.g., a LC variant having one or more amino acid substitutions, insertions, or deletions, and / or partial or whole domain substitutions, deletions, or insertions to modulate the duration of BoNT or TT action and / or potency.

[0075] In various aspects, the mRNA constructs can include one or more features to tailor the transfection efficiency. For instance, the mRNA constructs can include a 150 nucleotide poly(A) tail and / or a Cap 1 upgrade, in an aspect. In the same of alternative aspects, the mRNA QB\650053.01105\92299987.1 Page 19 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION constructs can be dsRNA reduced. In certain aspects, the mRNA constructs can be tailored to target neuronal cells. In alternative aspects, the mRNA constructs can be tailored to target other types of cells, including but not limited to cancer cells and / or cells associated with inflammatory processes.

[0076] In various aspects, the mRNA constructs can be formulated and / or present in lipid nanoparticles. In various aspects, the lipid nanoparticles can be any lipid nanoparticles suitable for use with the mRNA constructs and method disclosed herein. In one aspects, the lipid nanoparticles can be negatively charged, positively charged, or both.

[0077] In various aspects, compositions are disclosed herein. For instance, in certain aspects, the compositions can include one or more of the mRNA constructs disclosed herein. In certain aspects, the compositions can include one or of the mRNA constructs formulated and / or present in lipid nanoparticles. In alternative aspects, the compositions can include the 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 certain aspects, suitable pharmaceutically acceptable carriers can include RNAse-Free water, phosphate buffered saline, or the like.

[0078] The present disclosure also provides kits. In certain aspects, the kits can include a preparation of the BoNT variant, BoNT LC variant, mRNA constructs, or compositions disclosed herein, as well as instructions for administration. In one or more aspects, the kits can be used in the methods disclosed herein.

[0079] Methods

[0080] In various aspects, the methods disclosed herein can include a method of administering a BoNT variant or BoNT LC variant to a subject in need thereof. In certain aspects, the subject in need thereof can be a subject having a symptom in need of BoNT therapy. In various aspects, the methods disclosed herein can include treating a patient and / or subject with a therapeutically effective amount of a BoNT variant or BoNT LC variant. In such aspects, the treating can include administering a therapeutically effective amount of a BoNT variant or BoNT LC variant to the patient and / or subject. In another aspect, the treatment is a cosmetic treatment. It should be understood that the treatments contemplated by the present disclosure include not just treatments for effecting therapeutic action in neuronal cells, but for treatments in any type of cell, where inhibiting exocytosis may be desirable, including but not limited to inflammatory conditions, skin conditions such as acne, allergies, certain metabolic diseases, hormonal disorders, and cancer. In one aspect, the BoNT variants or BoNT LC variants can be administered for targeted treatment in cancer cells and / or cells associated with QB\650053.01105\92299987.1 Page 20 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION an inflammatory process. It should be understood that administering the BoNT LC variants can include administering an mRNA construct for producing the BoNT LC variant.

[0081] In certain aspects, the BoNT variant and / or BoNT LC variant can be administered in any suitable manner. In one or more aspects, the BoNT variant and / or BoNT LC variant can be administered topically or by injection, such as intramuscular or subcutaneous injection.

[0082] In various aspects, the BoNT variant and the BoNT LC variant can include any or all of the features discussed above with respect to the BoNT variants and BoNT LC variants.

[0083] In certain aspects, one or more of the mRNA constructs, formulations, and / or compositions can be administered to the patient and / or subject for one or more of the treatments mentioned above. In one or more aspects, the mRNA constructs can encode for one or more of the BoNT variants and / or BoNT LC variants disclosed herein. In certain aspects, the BoNT variant and / or BoNT LC variant can be administered as one or more mRNAs encoding for one or more of the BoNT variants and / or BoNT LC variants.

[0084] Timing and a dose of administration are not particularly limited and may vary depending upon severity of symptoms, age, sex, weight, site and route of administration. EXAMPLES

[0085] The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter.

[0086] Example 1 - Botulinum Neurotoxin Light Chain A1 usurps a synaptic vesicle cycling pathway to traffic to SNAP-25 on the plasma membrane of Neuro-2A cells

[0087] Summary

[0088] Botulinum neurotoxin serotype A (BoNT / A) is a potent neurotoxin but also a therapy to treat human neurological diseases. The 150 kDa BoNT / A contains a catalytic Light Chain (LC / A) linked by a disulfide bind to a Heavy Chain enabling entry into host cells. Molecular mechanisms involved in BoNT / A trafficking to the plasma membrane to target SNAP5-25 is an understudied aspect for BoNT intoxication. BoNT / A comprises eight subtypes (1-8), whereby previous data have shown distinct trafficking and intracellular localization for LC / A1 and LC / A3. This study utilized our knowledge of the roles of Rab proteins in synaptic vesicle recycling to further investigate LC / A trafficking inside the neuronal cell cytosol. Imaging studies colocalized LC / A1 with endogenous Rab GTPases and identified dominant negative-Rab GTPases that blocked LC / A1 plasma membrane localization, implicating a synaptic vesicle cycling pathway to traffic LC / A1 to the plasma membrane. Rab GTPases colocalized with intracellular LC / A3 were validated by Mass spectrometry. The QB\650053.01105\92299987.1 Page 21 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION identification of this synaptic vesicle cycling pathway to target LC / A1 to the plasma membrane identifies novel host proteins as targets for BoNT / A therapies to mitigate BoNT intracellular action.

[0089] Introduction

[0090] Clostridium botulinum are Gram-positive anaerobic bacteria that produce botulinum neurotoxins (BoNTs), the most toxic human proteins. BoNTs are 150 kDa zinc- dependent metalloprotease [1], possessing a 50-kDa N-terminal catalytic Light Chain (LC) and a 100-kDa C-terminal Heavy Chain (HC). The HC comprises a LC translocation domain (HCN) and a receptor binding domain (HCC), which enable neuronal cell association followed by endocytosis and translocation of the LC into the cell cytosol, respetivley [2-4]. BoNT includes seven pathogenic serotypes (A-G); BoNT / A includes eight subtypes (BoNT / A(1-8) [5, 8], which specifically cleave SNaptosomal-Associated Protein of 25 kDa (SNAP-25) on the cytoplasmic side of the neuronal plasma membrane [5].

[0091] LC / A delivery from the cell surface into the cytosol of neurons is well studied. BoNT / A binds a ganglioside on the surface of a resting neuron and subsequently binds a protein receptor, Synaptic Vesicle Protein-2 (SV2), exposed on the cell surface during SV exocytosis [6]. The BoNT / A-receptor complex is endocytosed during the recycling of the SV from the plasma membrane. SV mature and either fuse to the acidic endosome or directly undergo lumen acidification and neurotransmitter reloading. In response to acidification, BoNT / A HCNinserts into the SV membrane and facilitates LC translocation across the SV membrane into the neuronal cytosol [7]. Studies have indicated that inside the cell cytosol the disulfide bond between the LC and HC is reduced by the thioredoxin reductase system with the aid of HSP90, releasing the LC from into the cytosol (ref). However, molecular processes guiding intracellular LC / A1 to its enzymatic target, plasma membrane bound SNAP-25, is an understudied yet critical aspect of BoNT intoxication.

[0092] Recently, LCs of BoNT / A1, and BoNT / A3 subtypes were shown to possess different intracellular localizations where LC / A1 localized to the host cell plasma membrane and LC / A3 localized to intracellular vesicles. The higher potency of BoNT / A1 compared to BoNT / A3 raises the possibility that binding to the plasma membrane correlates with potency [8]. Studying LC / A1, LC / A3, and an N-terminal variant of LC / A3 termed LC / A3V, two regions within LC / A1 were shown to be responsible for LC / A1 intracellular trafficking to the plasma membrane, the N terminus (termed (A1 N), residues 1-17) which bound intercellular vesicles and an internal region of LC / A1 (termed the membrane localization domain (MLD), residues 275-334) which targeted a cytosolic target protein to the plasma membrane [8-11]. QB\650053.01105\92299987.1 Page 22 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION The current study further characterized pathways of intracellular trafficking by LC / A1, LC / A3, and LC / A3Vin Neuro-2A (N2A) cells by utilizing co-localization studies with a suite of Rab proteins. and the data show LC / A1 usurps a synaptic vesicle cycling pathway to target SNAP- 25 on the plasma membrane.

[0093] Results

[0094] Microtubules are involved in BoNT LC association with intracellular vesicles. In order to examine the role of cytoskeletal structures in LC / A1 intracellular trafficking, two cytoskeleton inhibitors were tested for effects on LC / A1 and LC / A3 intracellular localization in neuro-2a cells. Neuron 2a cells were first transfected with GFP (cytoplasmic), GFP-LC / A1 (membrane localized), GFP-LC / A3 (vesicle localized), or GFP-LC / A3V (cytoplasmic with nuclear exclusion) and allowed to express the fusion proteins before treatment with the cytoskeleton inhibitors. Cytochalasin D (CD) binds to the polymerizing ends of actin filaments, disrupting extension, inhibiting growth, and resulting in acting filament aggregation [12-17]. Phalloidin staining of CD-treated N2A cells showed actin disruption and ß-tubulin staining showed extensions of microtubule-stained neurites (FIG. 1A), but did not disrupt ectopically expressed EGFP-LC / A1 localization on the plasma membrane and LC / A3 localization on synaptic vesicles (SV). Time-lapse immunofluorescence imaging showed that in CD-treated N2A cells, EGFP-LC / A1 was membrane-localized and moved along neurites (FIG. 1B). Thus, EGFP-LC / A1 localizes at the plasma membrane and traffics along neurites independent of actin.

[0095] Nocodazole binds to ß-tubulin, preventing association with α-tubulin which depolymerizes microtubules, addressing the role of microtubules in LC / A1 trafficking [18, 19]. In nocodazole treated N2A cells, ectopically expressed EGFP-LC / A1 remained membrane- localized, while LC / A3 transitioned from a vesicle-associated protein to a cytosolic protein (FIG.1A), indicating nocodazole disrupted microtubules and dispersed LC / A3 into the cytosol. These studies implicate a role for microtubules in LC / A intracellular trafficking.

[0096] Colocalization of intracellular LC / A1 with Rab GTPases in N2A cells. Intracellular cargo trafficking utilizes Rab GTPases, which are compartmentalized to help stage vesicle transport within a cell

[0020] . Thus, Rab GTPases co-localization with LCs was characterized to determine the nature of vesicles associated with intracellular LC / A1. Initial experiments examined co-localization of Rab:LC / A1 in neuron-2a cells using Pearson's Correlation Coefficients (PCC)

[0021] . Colocalization determined by measuring the colocalization of an EGFP conjugated EGFP antibody with EGFP-LC / A1 (0.8 PCC) was set as maximum, while minimum colocalization was set as the EGFP-LC / A1 colocalization with QB\650053.01105\92299987.1 Page 23 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION Hoechst-stained DNA (-0.1 PCC) (FIG. 5). Steady-state ectopically expressed EGFP-LC / A1 showed the highest colocalization with Rab4, and Rab27a, lower colocalization with Rab5, Rab7, Rab11, and Rab4, and limited overall colocalization with Rab3a (FIG.2). Examinations of overlapping imaged pixels showed EGFP-LC / A1 colocalized with Rab3a and Rab27a along the plasma membrane, Rab4, Rab5, and Rab11 in the cell interior and Rab7 in the perinuclear region (FIG.2). Overall, the steady state colocalization data indicated LC / A1 trafficking was associated with Rab GTPases involved with several intracellular functions.

[0097] Dominant-Negative (DN) Rab GTPases block LC / A1 plasma-membrane localization. To identify Rab GTPases that regulate LC / A1 intracellular trafficking to the plasma membrane, DNA encoding individual EGFP-DN Rab GTPases were cotransfected with pDsRed-LC / A1, and the intracellular distribution of DsRed-LC / A1 was determined (FIG. 3). DN Rab GTPases preferentially bind GDP, sequestering Rho GEF and halting Rab-directed vesicle movement. Thus, DN Rab GTPases could identify Rab GTPases active in LC / A1 trafficking to the plasma membrane [22-24]. Cotransfection of EGFP-LC / A1 and DsRed- LC / A1 showed colocalization of the two fluorochromes. DsRed-LC / A1 was cotransfected with DNA-encoding DN Rab GTPases known to function in anterograde and retrograde trafficking to and from the plasma membrane [25-27], DN Rab3a, DN Rab4, DN Rab5, and DN Rab27b. DN Rab 3a, 4, 5m, and 27b localized cytosolic and sequestered LC / A1 from the plasma membrane (FIG. 3). Each positively correlated DN Rab localized LC / A1 differently within the cell. With coexpression of DN Rab3a or Rab5, LC / A1 was juxtaposed to the plasma membrane, while with coexpression of DN Rab4, or Rab27b, which functions upstream of Rab3

[0026] , LC / A1 was juxtaposed to the perinuclear region (FIG.3). Rab27a and Rab27b are isoforms

[0028] . In contrast, coexpression of neither DN Rab7 nor Rab11 altered LC / A1 plasma membrane localization, indicating that neither Rab7 nor Rab11 pathways were utilized for LC / A1 trafficking to the plasma membrane. Overall, the DN Rab GTPases that inhibited LC / A1 association with the plasma membrane function in the recycling SV pathway with the plasma membrane, implicating, for the first time, that LC / A1 utilizes the recycling SV pathway to anterograde traffic to the plasma membrane.

[0098] Control experiments tested the effect of the DN Rab GTPases in cholera toxin B subunit (CT / B) and transferrin endocytosis, as described earlier [29, 30] (FIG. 7). Although Rab4

[0031] and Rab11

[0032] participate in the trafficking of transferrin, expression of DN Rab4 or DN Rab11 did not inhibit transferrin and had limited effects on intracellular uptake and recycling of transferring in N2A cells (FIG.7), as seen with previous studies [33, 34]. QB\650053.01105\92299987.1 Page 24 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0099] Intracellular LC / A3 is associated with Rab GTPases involved with several vesicle trafficking pathways. To test for intracellular LC / A-Rab GTPases interactions, the association of Rab GTPases with ectopically expressed EGFP, EGFP-LC / A3, or EGFP-LC / A1 was measured by Mass Spectrometry (MS). N2A cells were lysed and post-nuclear supernatants (PNS) containing soluble proteins were prepared by centrifugation. MS analysis identified 29 Rab GTPases in the Post Nuclear Supernatant (PNS) of control (un-transfected) N2A cells (Supplemental Table 2-Table 3). Rab proteins associated with LC / A were identified by co-immunoprecipitation from the PNS and t LC-MS / MS. MS assessment showed a greater enrichment for Rab GTPases in the LC / A3-IP than the LC / A1-IP (Table 2), consistent with the steady state localization of LC / A3 bound to intracellular vesicles and LC / A1 primarily plasma membrane associated [8]. Pretreatment of the PNS with nocodazole prior to IPs increased recovery of LC / A3 and LC / A1, consistent with intracellular association on microtubules. Rab1a, Rab2(a / b), Rab3(a / c), Rab5b, Rab6a, Rab7a, Rab10, Rab11b, Rab14 and Rab21 were enriched > 2-fold in the co-IP of EGFP-LC / A3 relative to EGFP (Table 2). Rab4 and Rab27a are absent from co-IP of EGFP-LC / A3 or LC / A1, despite DN Rab4 and DN Rab27a inhibiting LC / A1 movement to the plasma membrane (Figure 3). Since the N2A proteome verified Rab4 and Rab27a present in the post nuclear supernatant, we concluded only a fraction of the total Rab4 and Rab27a interacted with LC / A3 or LC / A1 (Table 3). Overall, the Rab GTPases co- IPed with LC / A3 implicated LC / A interactions with the SV recycling pathway and late endosome and ER / Golgi trafficking. Table 2. Rab GTPases co-IPed with EGFP, EGFP-LC / A1, or EGFP-LC / A3LM in control or nocodazole-treated N2A cells. Table 2. Rab GTPases co-IPed with EGFP, EGFP-LC / A1, or EGFP- 6noitazilacoleQB\650053.01105\92299987.1 Page 25 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION Table 2. Rab GTPases co-IPed with EGFP, EGFP-LC / A1, or EGFP- LC / A3LM in control or nocodazole-treated N2A cells e e s utQB\650053.01105\92299987.1 Page 26 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION Table 3: Neuro-2A Rab Proteome

[0100] Discussion

[0101] The current study developed a transfection-based model to study the intracellular trafficking of ectopically expressed LC / A1 to the plasma membrane [8, 11]. Initial experiments implicated a role for microtubules in the trafficking of LC / A1 on intracellular vesicles. Next, QB\650053.01105\92299987.1 Page 27 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION steady-state imaging showed LC / A1 co-localized with several Rab GTPases that facilitated different cellular functions, including trafficking to the plasma membrane and protein degradation. These data were further confirmed by co-expression of LC / A1 with a subset of DN Rab GTPases, which implicated the SV cycling pathway in LC / A1 trafficking to the plasma membrane. Finally, co-immunoprecipitation of intracellular LC / A1 and LC / A3. Coupled to MS assessment of co-IPed proteins provided a global assessment of Rab GTPase-LC / A1 interactions and revealed association with Rab GTPases involved in the SV cycling pathway as well as in the intracellular degradation pathways. This is the first determination of an intracellular vesicle pathway for the trafficking of LC / A to stably co-localize with SNAP-25 on the plasma membrane, indicating LC / A1 trafficking via both anterograde- and retrograde- pathways that lead to SNAP-25 targeting and LC degradation

[0045] . This approach can be used to measure the trafficking of the catalytic domain of other protein toxins to their intracellular substrates, an understudied aspect of protein toxin action.

[0102] LC / A1 remained membrane localized in the presence of nocodazole and cytochalasin D, while LC / A3 diffused into the cytosol in nocodazole-treated N2A cells (FIG. 1A). The effect of nocodazole on LC / A3 but not LC / A1 is likely due to LC / A1 at steady state already being stably associated with the intracellular plasma membrane and SNAP-25, whereas LC / A3 is predominantly vesicle and cytosol localized and thus trafficking to the plasma membrane can still be interrupted. Thus, it is likely that LC / A1 and LC / A in general also utilizes microtubules for intracellular trafficking to the plasma membrane. Microtubules play a critical role in numerous cellular activities including cytokinesis, transport, and motility. As such, microtubules are often exploited by bacterial toxins. Our studies examined steady state levels of intracellularly expressed LC / A, whereas the natural intoxication pathway of neurons by BoNTs involves a multistep entry process. BoNT / A binds to gangliosides and an SV protein on the surface of a neuron prior to endocytosis [35, 36]. BoNTs enter cells in the lumen of vesicles primarily through clathrin-dependent pathways [37-39]. Studies on BoNT-HC entry into N2A cells showed that treatment with nocodazole decreased HC binding, while genistein, an inhibitor of caveolae-mediated endocytosis and actin networks, did not reduce HC binding [40, 41]. Combined with our data, these studies show that BoNT / A utilizes microtubules for entry and intracellular trafficking. In N2A cells treated with cytochalasin D, which results in actin filament aggregation, time-lapse imaging showed LC / A1 moving along microtubules in neurite sproutes (FIG.1B).

[0103] While previous experiments characterized the contribution of Rab GTPases during BoNT intoxication [42-44], the current study measured the intracellular trafficking of QB\650053.01105\92299987.1 Page 28 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION ectopically expressed EGFP-LC / A1 relative to colocalization with endogenous Rab proteins and in the presence of DN Rab proteins, and finally examined direct interactions of endogenous Rab GTPases by co-IP experiments with LC / A1 and / A3, implicating a synaptic vesicle cycling pathway to traffic LC / A1 to the plasma membrane (FIG.3).

[0104] Several Rab GTPases co-localized with LC / A1 and several DN Rab GTPases inhibited LC / A1 trafficking to the plasma membrane. DN Rab5 inhibited LC / A1 trafficking to the plasma membrane, sequestering LC / A1 on intracellular vesicles (FIG. 3). Rab5(a-c)

[0045] mediate clathrin-dependent endocytosis, a major pathway for SV retrieval from the plasma membrane [27, 46, 47]. In Drosophila, DN Rab5 mediated the homotypic fusion of SVs, increasing SV size and inhibiting neurotransmitter release

[0048] . Furthermore, DN Rab5 inhibits early endosome fusion

[0049] . As observed with ExoS

[0050] , DN Rab5 did not localize with LC / A1 whereas Rab5 showed moderate co-localization, indicating DN Rab5 blocked the formation of the sorting endosome, either trapping bound LC / A1 upstream of the sorting endosome or the lack of sorting endosomes prevents the trafficking to the recycling endosome. LC / A1 showed a higher degree of co-localization with Rab4 than with Rab 11, and DN Rab4, but not DN Rab11, inhibited LC / A1 trafficking to the plasma membrane (FIG. 3). Rab4 and Rab11 facilitate endosomal recycling prior to the formation of SVs

[0051] . Kinesin-2-mediated endosome recycling is categorized into two populations, Rab4-mediates fast recycling and Rab4 to Rab11-mediates slow recycling [51-53]. Thus, LC / A1 may usurp the Rab4-mediated fast recycling route to reach the plasma membrane. Further, a high degree of co-localization of LC / A1 was observed with Rab27b, whereas little co-localization was seen with Rab3a (Figure 2). Yet both, DN Rab3a or DN Rab27b expression inhibited LC / A1 trafficking to the plasma membrane (FIG.3). In neurons, Rab3a and Rab27b control SV docking, fusion, and exocytosis

[0051] . Rab3(a-d)

[0054] is pivotal for fast Kinesin1-mediated anterograde trafficking and vesicle assembly on microtubules

[0055] . While Rab3(b-d) are redundant, KO models showed that the removal of Rab3a was lethal post-birth

[0056] . Rab3 and Rab27(a-b)

[0028] are structurally related, partially colocalized, and share effector proteins such as Rabphilin3a, which binds to Rab3 and Rab27 and regulates SV cycling prior to trafficking and localization at the plasma membrane with SNAP-25 [57, 58]. [59-61]. While present in overlapping pools, temporally Rab3 and Rab27 transition distinct SVs

[0057] , which could explain the inhibition of LC / A1 trafficking to the plasma membrane upon expression of either DN Rab3a or DN Rab27b yet greater co- localization with Rab27b. DN Rab7 did not interfere with LC / A1 trafficking to the plasma membrane (FIG. 3). Association of LC / A1 with Rab7 and other Rab GTPases involved in retrograde protein trafficking may reflect a common interaction between LC / A1 and QB\650053.01105\92299987.1 Page 29 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION intracellular vesicles involved in both retrograde and the anterograde SV recycling pathway that delivers LC / A1 to the plasma membrane (Table 2).

[0105] Utilizing data from the current study, studies on Rab GTPases action [26, 27], and previous data on BoNT trafficking and LC / A localization [8, 40, 62], we propose a model of BoNT / A intracellular trafficking (FIG. 4). Initially, BoNT / A1 binds a gangliosides and activity-dependent surface exposed SV-2. Bound BoNT / A1 is internalized into early endosomes (Rab5) and retrogradely trafficked along microtubules to the perinuclear region where maturation of SV recycling endosomes occurs, resulting in luminal acidification and LC translocation across the vesicle membrane. In the cytosol, LC / A1 is released via disulfide bond reduction, refolds, and associates with Rab4-positive SVs via the N terminus (N) for anterograde trafficking along microtubules transitioning to Rab3a-Rab27b vesicles. When juxtaposed to the plasma membrane, LC / A1-MLD stably binds SNAP-25 for cleavage.

[0106] While single DN Rab GTPases often implicate the use of a signaling pathway

[0063] , Rab GTPases can have specialized functions in neurons

[0027] . The current study used three approaches to define and validate multiple Rab GTPases that regulate a known synaptic vesicle trafficking pathway for LC / A1 early endosome interactions to the stable association with SNAP-25 on the plasma membrane and implicate the dynamic nature of LC / A intracellular trafficking for potential degradation. Understanding the dynamics of BoNT LC intracellular trafficking may allow targeting of host proteins to mitigate BoNT toxicity and design of novel pharmaceuticals.

[0107] Materials and Methods

[0108] Reagents were purchased from Life Technologies (Grand Island, NY, USA) unless otherwise specified.

[0109] Engineering of EGFP-LC / A Constructs and Rab Dominant Negative. Plasmid pEGFP-C3 encoded DNA from A1 (1-4450), A3V (1-446), and A3LM (1-446) was engineered as described previously [8]. Enhanced green fluorescent proteins (EGFP) fusions with LC / A1, LC / A3V, LC / A3LM, and Rab GTPases 3a, 4, 5, 6, 11, and 27a were subcloned into the SacI- BamHI restriction sites of pEGFP-C3. Primers were designed and obtained from New England Biolabs® NEBaseChanger® (Ipswich, MA, USA) to engineer dominant negative variants of the Rab proteins: Rab3a (T36N), Rab4 (S22N), Rab5 (S34N), Rab7 (T22N), Rab11 (S25N), and Rab27b (T23N).

[0110] Cell Culture. Neuro-2A (N2A) cells were from ATCC (Manassas, VA, USA) CCL-131. N2A cells were cultured in complete essential media supplemented with 10% fetal QB\650053.01105\92299987.1 Page 30 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION bovine serum, 1x penicillin-streptomycin, 0.1% sodium bicarbonate, 1mM sodium pyruvate, and 1% nonessential amino acids in humidified 5% CO2(v / v) at 37 °C.

[0111] Transfection and cytoskeleton inhibitor treatment of N2A cells. N2A cells were transfected as previously described

[0064] . N2A cells were seeded in a 24-well plate at 50,000 cells / well on acid-etched glass coverslips coated with Poly-D lysine (1:500). The following day, N2A cells were transfected (Lipofectamine LTX; Invitrogen™ (Waltham, MA, USA)) with 500ng of the indicated plasmid, as described earlier [8]. Following an overnight transfection, cells were treated with 4µM of Cytochalasin D (C8273, Sigma-Aldrich, St. Louis, USA), an actin inhibitor, or 9µM of nocodazole (M1404, Sigma-Aldrich, St. Louis, USA), a microtubule inhibitor, for 60 min.

[0112] Immunofluorescence Imaging (IF). After transfection, N2A cells were fixed with 4% (w / v) paraformaldehyde for 15 min at RT. N2A cells were then washed 2x with RT PBS and blocked for 60 min in 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 DBPS (14040182, Gibco™, IL, USA). N2A cells were incubated with primary antibodies: 1:5000 rabbit α-ß-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 (ab 137029, Abcam, Cambridge, UK), 1:1000 rabbit α-Rab11 (ab180504, Abcam, Cambridge, UK), or 1:1000 rabbit α-Rab27a (ab223044, Abcam, Cambridge, UK) shaking, overnight at 4°C in 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) in DBPS (incubation solution). The following day, N2A cells were washed 3x (five min) with 0.005% Tween-20 (v / v) in DPBS and incubated with complimentary Alexa-Fluor-conjugated secondary or phalloidin-Alexa- fluor 568 nm (1:250) (A12380, Life Technologies, WI, USA) in incubation solution for 60 min at RT, washed 2x in 0.005% Tween-20 (v / v) DPBS, and stained with Hoechst (1:10,000 in DPBS, H21492, Invitrogen™, MA, USA) for 5 min. Coverslips were flipped onto microscope slides and cured with 8µL of Prolong™ Gold Antifade Mountant (Invitrogen, MA, USA).

[0113] Additionally, N2A cells were washed 2x times with 4°C DPBS and stained with wheat germ agglutinin (WGA) (1:1000, W32466, Invitrogen, MA, USA) as a membrane stain for 30 min at 4°C. Following WGA incubation, N2A cells were washed 2x with DPBS and then fixed with 4% (w / v) paraformaldehyde for 15 min at RT, washed 2x with DPBS, and treatment Hoechst (1:10,000 in DPBS, H21492, Invitrogen™, MA, USA) as a nuclear marker for 5 min at RT. Coverslips were flipped onto microscope slides and cured with 8µL of Prolong™ Gold Antifade Mountant (Invitrogen, Waltham, MA, USA). QB\650053.01105\92299987.1 Page 31 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0114] Live-cell imaging. N2A cells were cultured and analyzed as previously described [8, 64]. Four h post-transfection with LC / A1, as described above, N2A cells were treated and incubated for 60 min with 4µM of cytochalasin D. N2A cells were live-imaged on a Nikon Eclipse Ti-inverted microscope using a 60x oil-immersion objective (1.4 NA objective) with a heated stage at 37°C (Frank E. Fryer A-50) with Eclipse software for data analysis. Images were acquired every 15 seconds for 15 min. The video was compiled utilizing Nikon Elements AR 1.60.0064-bit software (Melville, NY, USA) and Image J [65, 66].

[0115] EGFP-LC / A Coimmunoprecipitation. N2A cells were seeded at 250,000 cells / well in a six-well plate coated with Poly-D lysine (1:500). N2A cells were transfected (Lipofectamine LTX; Invitrogen™ (Waltham, MA, USA)) with 3.0µg of indicated plasmid DNA. After overnight transfection, N2A cells were washed twice at 4°C with DPBS in a precooled centrifuge tube. N2A cells were pelleted at 500x g for 3 min at 4°C, the supernatant was discarded, and cells were washed 2x in 4°C DPBS. N2A cells were resuspended in 200µL of lysis buffer: 10mM Tris / Cl pH 7.5; 150mM NaCl; 0.5mM EDTA; supplemented with 1:100 protease inhibitor and 1mM PMSF and cells were lysed by 30 passages through a 30-G needle. The cellular lysate was centrifuged at 20,000x g for 10 min at 4°C; the soluble Post-Nuclear Supernatant (PNS) was transferred to EGFP-TRAP®_MA beads (proteintech® Rosemont, IL, USA) equilibrated in dilution / wash buffer 10mM Tris / Cl pH 7.5; 150mM NaCl; 0.5mM EDTA, supplemented with 1:100 protease inhibitor, and 1mM PMSF and incubated at 4°C for 60 min. EGFP-TRAP®_MA beads were magnetically separated and washed 3x in wash buffer. Immunocomplexes bound to EGFP-TRAP®_MA beads were dissociated after 95°C boiling for 10 min in a 2x Protein sample buffer (PSB).

[0116] Western Blot. EGFP-TRAP®_MA elution was collected in 2x PBS and subjected to SDS-PAGE GEL as previously described [8]. Samples were resolved on a 13.5% SDS- PAGE and transferred to an Immobilon-P polyvinylidene difluoride membrane (PVDF) (Millipore, Billerica, MA, USA). PVDF membranes were rehydrated in methanol, washed in reverse osmosis water, stained with Ponceau (P3504, Sigma-Aldrich, St. Louis, USA), and incubated in blocking solution (2% powder milk (w / v) in 0.1% TBST). PVDF membranes were incubated with 1:2000 rat α-EGFP-monoclonal IgG (3H9, Chromotek, Planegg, DE), 1:20000 mouse α-ß-actin monoclonal IgG (A2228, Millipore Sigma, Mass, USA), or 1:2000 rabbit α-ß-3 tubulin (ab3335, Abcam, Cambridge, UK) shaking, overnight, at 4°C. Bound primary antibodies were recognized as 1:10,000 α-rat, 1:20000 α-mouse, or 1:10,000 α-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (Life Technologies, MA, USA). Secondary antibodies were visualized using Super Signal™ West Pico PLUS QB\650053.01105\92299987.1 Page 32 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION Chemiluminescent Substrate (34578, Thermo, IL, USA) on an Azure C600 Imaging System (Dublin, CA, USA), using a 60-second exposure.

[0117] Mass spectrometric assessment of Rab GTPases associated with LC / A A1 and LC / A3LM. EGFP-TRAP®_MA elution was collected in clean 2x PBS and resolved in a gradient (8-16%), stain-free protein gels (5678104, Bio-Rad, Hercules, California, USA). The samples were resolved ~1 centimeter into the gel, far enough to discern the 250 kDa molecular weight marker. The gels were then fixed in 30% isopropanol and 10% glacial acetic acid for 30 min, then washed 3x for 10 min each in HPLC grade water (EM-WX0008-1, VWR, PA, USA). Entire samples were excised from the gel and packaged individually for analysis by mass spectrometry at the Taplin Biological Mass Spectrometry Facility at Harvard Medical School. Mass spectrometry results were received as a compiled list in an Excel spreadsheet. The Excel spreadsheet included the Uniprot code, gene symbol, annotation, molecular weight, and total and unique peptides detected. The samples were analyzed in an unbiased, then biased manner. The biased analysis focused on enriched proteins 2-fold over EGFP, while the unbiased comprised the entire data set. The MS-derived N2A proteome used in this study identified over 3500 proteins present in the total cell lysate, soluble fraction, and(or) the membrane "pelleted" fraction was attached (Supplemental Figure 4b). Table 3: The number of proteins (unbiased and biased1) identified by mass spectrometry- untreated and nocodazole treated Neuro-2a Cells Table 3. The number of proteins (unbiased and biased1) identified by mass s ectrometr - untreated and nocodazole treated Neuro-2a Cells ,

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[0184] 66. Schindelin, J., et al., Fiji: an open-source platform for biological-image analysis. Nat Methods, 2012.9(7): p.676-82. Example 2: The N-terminus plays only a minor role in duration of action of BoNT / A

[0185] Introduction

[0186] During our studies we identified two aa alterations near the N-terminus (Q7P, V14G) of the BoNT / A LC that convert the punctate (vesicle associated) intracellular localization of BoNT / A3 to a diffuse intracellular localization. This indicates the BoNT / A N- terminus is involved in intracellular LC trafficking by conveying synaptic vesicle association. To examine this further, we created a recombinant BoNT / A3 holotoxin containing these two aa alterations, purified the holotoxin, and examined its duration of action in primary neuronal cultures and in mice.

[0187] Methods

[0188] Generation of a recombinant gene for expression of BoNT / A3 Q7P, V14G. BoNT / A3 gene was amplified by PCR using total genomic DNA isolated from C. botulinum subtype A3 strain (kindly provided by the CDC) and Fusion Hot Start Flex 2x Master mix according to manufacturer’s instructions (New England Biolabs). To introduce two amino acid mutations into the N-terminus of the toxin, two nucleotide substitutions were included into the 5’ PCR primer: Mutation Gln7to Pro (CAA to CCA) and Val14to Gly (GTA to GGA). Primers A3-Nde-5’ and A3-Sal-3’ were used to amplify bont / A3 mutant gene. A3-Nde-5’ GGCATATGCCATTTGTTAATAAACCATTTAATTATAGAGATCCTGGAAATGGTG (SEQ ID NO: 1). A3-SalI-3’ GCGTCGACCTTACAGTGAACTTTCTCCCCATCCATCATC (SEQ ID NO: 2).

[0189] The nucleotide sequence of the mutated recombinant toxin gene was verified by DNA sequencing. Then the recombinant gene was inserted into modular clostridial expression vectors pMTL82152 and pMTL83152 [1]. Recombinant expression vectors were transferred QB\650053.01105\92299987.1 Page 38 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION into nontoxigenic C. botulinum expression host strain Hall A-hyper / tox- by conjugation from E. coli donor strain CA434, and expression of the recombinant mutant toxin evaluated as previously described [2].

[0190] Production of BoNT / A3 Q7P, V14G. Recombinant BoNT / A3-2M (Q7P, V14G) was expressed in the atoxic C. botulinum strain Hall A- hyper / tox- [2] using the pMTL82152 vector [1].

[0191] The rBoNT / A3-2M was purified according to the method previously described for purification of BoNT / A1 [3], with an additional chromatography step after the DEAE, pH 8.0 column to remove complex proteins on a 1.5 ml p-aminobenzyl 1-thio-b-D- galactopyranoside (pABTG) agarose affinity column as previously described [4]. Purified toxin was passed through a 0.2 mm filter and mixed with an equal volume of 80% sterile glycerol and stored at -20oC.

[0192] The purified BoNT / A3-2M was analyzed by 4-12% Bis-Tris (Novex NuPAGE, Life Technologies) gel electrophoresis either non-reduced or reduced with 100 mM DTT, and stained with InstantBlue (Expedeon), and concentration and purity were determined by spectroscopy (absorbance at 278 nm) and densitometry.

[0193] Mouse Bioassay. Activity of the purified 2M-rA3 was determined using a standard intraperitoneal mouse bioassay (MBA) as previously described [3,5,6]. Briefly, BoNT / A3-2M was diluted to a final concentration of 44, 88, 134, 178, 266, and 356 pg / ml in GelPhosphate buffer (0.03M sodium phosphate, pH 6.3, 0.2% gelatin). Groups of 4 female ICR mice (18-22 g) were injected intraperitoneally (i.p.) with 0.5 ml of each dilution and observed for 4 days for signs of botulism and death. The specific activity was calculated according to the method of Reed and Muench [7].

[0194] Cell based assay. Primary rat spinal cord (RSC) cells were prepared from E15 Sprague Dawley rat pups and seeded into 96-well TPP plates that had been coated with 0.01% poly-L-ornithine and 8.3 µg / cm2 Matrigel at a density of 50,000 cells / well. RSC cells were maintained in Neurobasal media supplemented with B27, glutamax, and penicillin / streptomycin (all from Life Technologies) as previously described [8,9] and used for the toxin assay after 19 days in culture. Cells were exposed to either 15 mouse LD50 Units (mLD50) of BoNT / A3 wt or 15 mLD50 of BoNT / A3- Q7P, V14G for 48 h, then all extracellular toxin was washed off and cells were incubated in fresh culture media with bi- weekly media changes. Triplicate samples of the cells were harvested at the indicated time points in 1 x LDS samples buffer (Life Technologies), and cell lysates were analyzed by Western blot and densitometry for the percentage of SNAP-25 that had been cleaved by BoNT QB\650053.01105\92299987.1 Page 39 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION using a monoclonal anti-SNAP-25 antibody that recognizes both cleaved and uncleaved SNAP-25 (Synaptic Systems, Germany) as previously described ([8,9]). Images were obtained using PhosphaGlo reagent (KPL, Gaithersburg, MD) and an Azure C600 Imaging system. The percent un-cleaved SNAP-25 reflects recovery of the cells from the BoNT intoxication.

[0195] Results

[0196] The N-terminus plays only a minor role in duration of action of BoNT / A3. SDS-PAGE analysis of the purified recombinant BoNT / A3- Q7P, V14G 2M showed a single 150 kDa band in the non-reduced sample, and in the reduced sample, more than 90% of the toxin was observed as the 100 kDa and 50 kDa LC and HC bands, respectively, indicating >90 % dichain toxin (FIG.8).

[0197] A standard mouse bioassay determined that the specific activity of the purified 2M- rA3 toxin was ~1 x 107LD50 / mg (~100 pg / LD50). This compares to a specific activity of ~5.8 x 107LD50 / mg (~17 pg / LD50) for wt BoNT / A3 [10,11].

[0198] To determine whether the N-terminus affects duration of action, mice were locally injected with either BoNT / A3 wt or BoNT / A3- Q7P, V14G into the gastrocnemius muscle, and Rotarod assay and digital abduction score (DAS) assay were conducted to determine motor-neuron deficiency and local paralysis, respectively. While little overall motorneuron deficiency was detected with either toxin, which is consistent with our previous data, a maximal DAS score of about 4 was reached with both toxins (FIG. 9). There was little difference in recovery from the DAS score between BoNT / A3 and the mutant BoNT / A3 - Q7P, V14G, indicating little or no effect of the N-terminal mutations on duration of action.

[0199] For further analysis of the duration of action, primary rat spinal cord cells were exposed to either BoNT / A3 wt or BoNT / A3- Q7P, V14G, and the recovery from SNAP-25 cleavage was monitored over time. There was little difference in the recovery rate of SNAP- 25 cleavage for the cells exposed to BoNT / A3 wt versus BoNT / A3- Q7P, V14G (FIG.10).

[0200] Conclusion

[0201] Taken together these data indicate that the N-terminus of the BoNT / A3 LC has no or little effect on duration of action. Example 3: Functional BoNT / A LC can be introduced into human neurons via mRNA transfection leading to long lasting duration of action determined by the LHD domain.

[0202] Introduction

[0203] The use of mRNA as therapeutics has been established for vaccines. In that case, mRNA is packaged in lipid-nanoparticles and delivered to host cells, which then produce the antigen and release it into the circulatory system, where antibodies are made against the QB\650053.01105\92299987.1 Page 40 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION antigen. However, mRNA can also be used to produce a desired protein within a target cell without introducing DNA material into the cell, which might interact with the host genome. The challenge with this approach is the successful delivery of the mRNA into a sufficient number of target cells to achieve a therapeutic effect. In this example, human (iPSC derived) neurons were targeted with BoNT-LC mRNA. Neurons, in particular hiPSC derived neurons, are difficult to transfect with either DNA or mRNA, thus potentially precluding the therapeutic utility of BoNT LC mRNA. However, as we show in this example, we have been successful in achieving high transfection efficiency (up to 90%) and measurably long lasting inhibition of exocytosis in human neurons. As the N-terminus of the BoNT / A LC did not appear to determine duration of action, we investigated the effect of the LHD using a novel approach in which we transfected human induced pluripotent stem cell (hiPSC) derived motor-neurons (provided by BrainXCell) with synthesized mRNA constructs encoding the BoNT / A LC and derivatives. For this experiment, several hurdles had to be overcome. We had to create an mRNA construct that evades cellular immune defenses within the neuronal cell, is expressed into a functional and properly folded LC within the cell, and we had to devise an optimal cell transfection protocol for hiPSC derived neurons, which are notoriously difficult to transfect. Initial attempts using mRNA prepared in the lab using standard nucleotide modifications and capping methods resulted in weak transfection efficiency (15-30%), even after transfection of the neurons was optimized.

[0204] Methods

[0205] Neuronal Cell Culture. Spinal motor neurons derived from human induced pluripotent stem cells (hiPSCs) were provided by BrainXell (Madison, WI) and used essentially as previously described

[0012] . The cells were seeded into 96-well TPP plates (Techno Plastic Products, Midwest Scientific, Valley Park, MO) that had been coated with 0.01% poly-L- ornithine and 8.3 µg / cm2 Matrigel (BD Biosciences, East Rutherford, NJ) at a density of about 38,000 cells per well and maintained as per manufacturer’s instructions. Recommended culture media was used, comprised of equal parts Neurobasal and DMEM / F12 Medium supplemented with B27, N2 and GlutaMAX (Life Technologies). Cells were cultured for 3 days prior to transfection with mRNA and for 7 days prior transfection with LC / A1 mRNA in LNP formulation.

[0206] mRNA Constructs. All mRNA constructs were purchased from RiboPro (Netherlands) and were produced with the following modifications: ^ RNA synthesis from de novo ^ template / plasmid QB\650053.01105\92299987.1 Page 41 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION ^ dsRNA reduced ^ Cap1 upgrade ^ 150nt Poly-A tail ^ Buffer: RNAse-Free-Water ^ Concentration: 1 μg / μl

[0207] For the lipid formulation BoNT / A1 LC mRNA, production was as follows: ^ RNA synthesis from de novo ^ template / plasmid ^ dsRNA reduced ^ Cap1 upgrade ^ 150nt Poly-A tail ^ Formulated in ciLNP in PBS ^ Concentration: 100 ng / μl

[0208] The following constructs were created: LC / A1 aa1-450, LC / A3 aa 1-446, 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 sequences and nucleotide sequences are provided below.

[0209] Lipofectamine MessengerMax Transfection. Cells were transfected using Lipofectamine MessengerMax reagent (Invitrogen) in OptiMEM Reduced Serum Medium (Gibco). Lipofectamine complexes were prepared by diluting Lipofectamine MessangerMax reagent into OptiMEM media in a 3:100 volume ratio. The Mixture was incubated for 10 minutes at room temperature. Custom LC / A1 and A3 mRNA constructs were diluted in OptiMEM media to a concentration of 40 ng / ^l. Equal volumes Lipofectamine MessengerMax solution and mRNA dilutions were combined and incubated for 5 minutes at room temperature. 10 ^l complexes were added to cells containing final amounts of 0.15 ^l Lipofectamine MessengerMax reagent and 200 ng mRNA per well. Cells were incubated for 24 hours at 37 ^C and 5% CO2 before removing the complexes and washing cells with culture media. Samples of cells (n=5) were collected about every 10 days starting at day 6 post transfection in 1 x NuPAGE LDS Sample Buffer and heated at 95 ^C for 10 minutes. Samples were stored at - 20 ^C for Western Blot analysis.

[0210] RiboPro LNP formulation Transfection. 7 Days after seeding hiPSC-derived motor neurons, the cells were transfected with LC / A1 mRNA in RiboPro’s lipid nanoparticle formulation. 100 or 200 ng LC / A1 mRNA in LNP per well was added directly to the cells or was first diluted in OptiMEM media before added to cells. Cells were incubated with complexes at 37 ^C and 5% CO2 and samples were collected in 1 x NuPAGE LDS Sample QB\650053.01105\92299987.1 Page 42 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION Buffer after 24 and 42 hours. Samples were heated at 95 ^C for 10 minutes and store at -20 ^C for Western Blot analysis.

[0211] Western Blot Analysis. Cell lysates were analyzed by Western blot and densitometry for the percentage of SNAP-25 that had been cleaved using a monoclonal anti- SNAP-25 antibody that recognizes both cleaved and uncleaved SNAP-25 (Synaptic Systems, Germany) as previously described ([8,9]). Images were obtained using PhosphaGlo reagent (KPL, Gaithersburg, MD) and an Azure C600 Imaging system. Densitometry was conducted using the Azure spot software version 2.0.062, and average and standard deviations of the five replicates were used to create a graph of cleaved SNAP-25 (in percent compared to un-cleaved) versus time in excel. The data points were fitted with logarithmic trendlines.

[0212] Results

[0213] BoNT / A LC mRNA transfection of neuronal cells results in functional intra- neuronal LC, with duration of action determined by LHD. Transfection of the motor- neurons with a GFP control mRNA showed about 70% of cells had been transfected. For the BoNT / A LC constructs, transfection efficiency was evaluated by percent cleaved SNAP-25 at the first time point 6 days post transfection. At this time point, percent SNAP-25 cleavage ranged from 11 % to over 60%, with BoNT / A1 LC resulting in a mean of 40 ±12 % cleaved SNAP-25 (FIG.11). This indicates excellent transfection efficiency, with the lower values of some of the constructs correlating to lower LC potency previously determined for holotoxins (see Example 2). Decrease of the percent cleaved SNAP-25 was monitored up to 60 days post transfection at approximately 10 – 14 day time intervals. For all LC constructs, a logarithmic decrease in percent cleaved SNAP-25 was observed, which was most rapid for the LC / A3- Q7P, V14G and LC / A1 (A3LHD). A similar rate of rapid decrease of LC activity was observed for LC / A3, although this LC started at a higher rate of cleavage due to greater potency as compared to the LC / A3-Q7P, V14G, and thus cleaved SNAP-25 was observed for a longer total time period. This is consistent with the dose-dependent effect in duration of action known to occur with BoNTs. The LC / A1 as well as LC / A3 (A1LHD) showed a much slower decrease in cleaved SAP-25, indicating longer persistence of LC activity inside the motor-neurons for LC / A1 and LC / A3 containing the A1 LHD.

[0214] RiboPro LNP formulation of LC / A1 mRNA results in almost complete transfection efficiency in human motor-neurons. In order to examine whether the lipid formulation offered by RiboPro may further enhance transfection efficiency, hiPSC derived motor-neuron were transfected with lipid-formulated LC / A1, and cell lysates were analyzed QB\650053.01105\92299987.1 Page 43 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION after 24 h or 42 h for percent cleaved SNAP-25. Depending on the mRNA amount used and incubation time with the cells, a maximum of ~ 85% cleaved SNAP-25 was achieved, which indicates greater than 80% transfection efficiency (FIG.12).

[0215] Conclusions

[0216] These data demonstrate successful transfection of human neurons with BoNT / A LC, resulting in production of functionally active LC inside the targeted neurons. LC activity persisted for a prolonged time consistent with activity observed in hiPSC derived neurons intoxicated with BoNT / A protein. The data further show that the low homology domain (LHD) is responsible for the long duration of action of BoNT / A LC. Finally, we demonstrate that even cells that are notoriously difficult to transfect, such as hiPSC derived neurons, can be very efficiently transfected with mRNA formulated in nanoparticles. Taken together these data indicate feasibility of utilizing BoNT LC mRNA constructs therapeutically as an alternative to protein based therapy. Further, the duration of action can be customized through sequence modification of the mRNA in the MLD domain. Since nanoparticle targeting is not restricted to neurons, additional targets including inflammatory cells or cancer cells are feasible targets for such a therapy.

[0217] Amino Acid and Nucleotide Sequences for mRNA Constructs:

[0218] WT LC / A1

[0219] Amino acid sequence (SEQ ID NO:3):

[0220] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRML LTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIH AGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYY YNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEI YTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQN TEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL-

[0221] DNA sequence (SEQ ID NO:4):

[0222] ATGCCGTTTGTGAACAAACAGTTTAACTATAAAGATCCGGTGAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCAGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG QB\650053.01105\92299987.1 Page 44 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGACCAAACTGTTTGAACGTATTTATAGCACCGATCTGGGCCGTATGC TGCTGACCAGCATTGTGCGTGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTCAGCCGGATGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGAAGTGCTGAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCAAATTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGGCCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGTTTAAAGTGAACACCAACGCGTATTATGAAA TGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCCATGATGCG AAATTTATTGATAGCCTCCAGGAAAACGAATTTCGTCTGTATTATTATAACAAATTTAAAGA TATTGCGAGCACCCTGAACAAAGCGAAAAGCATTGTGGGCACCACCGCGAGCCTCCAGTATA TGAAAAACGTGTTTAAAGAAAAATATCTGCTGAGCGAAGATACCAGCGGCAAATTTAGCGTG GATAAACTGAAATTTGATAAACTGTATAAAATGCTGACCGAAATTTATACCGAAGATAACTT TGTGAAATTTTTTAAAGTGCTGAACAGAAAAACCTATCTGAACTTTGATAAAGCGG TGTTTAAAATTAACATTGTGCCGAAAGTGAACTATACCATTTATGATGGCTTTAA CCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGCCAGAACACCGAAATTAA CAACATGAACTTTACCAAACTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAA CTGCTGTGTGTGCGTGGCATTATTACCAGCAAAACCAAAAGCCTGGATAAAGGC TATAACAAAGCGCTGTAA

[0223] WT LC / A3

[0224] Amino acid sequence (SEQ ID NO: 5):

[0225] MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRML LSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSF GHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELI HAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSR DAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLT RGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEIN SRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0226] DNA sequence (SEQ ID NO: 6): QB\650053.01105\92299987.1 Page 45 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0227] ATGCCGTTTGTGAACAAACAATTTAACTATCGTGATCCGGTAAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGATAC CAACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATAACCTGCAGA ACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCACCACCCCGCTGCAGTAT ATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGAAGATGCGAGCGGCAAAATTAGCGT GAACAAAGCGGCGTTTAAAGAATTTTATCGTGTGCTGACCCGTGGCTTTACCGAACTGGAAT TTGTGAACCCGTTTAAAGTGATTAACCGTAAAACCTATCTGAACTTTGATAAAGCG GTGTTTCGTATTAACATTGTGCCGGATGAAAACTATACCATTAACGAAGGCTTTA ACCTGGAAGGCGCGAACAGCAACGGCCAGAACACCGAAATTAACAGCCGTAACT TTACCCGTCTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTG CGTGGCATTATTCCGTTTAAAACCAAAAGCCTGGATGAAGGCTATAACAAAGCG CTGTAA

[0228] JV LC / A3

[0229] Amino Acid sequence (SEQ ID NO: 7):

[0230] MPFVNKPFNYRDPGNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRML LSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSF GHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELI HAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSR QB\650053.01105\92299987.1 Page 46 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION DAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLT RGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEIN SRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0231] DNA sequence (SEQ ID NO: 8):

[0232] ATGCCGTTTGTGAACAAACCGTTTAACTATCGTGATCCGGGCAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGATAC CAACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATAACCTGCAGA ACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCACCACCCCGCTGCAGTAT ATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGAAGATGCGAGCGGCAAAATTAGCGT GAACAAAGCGGCGTTTAAAGAATTTTATCGTGTGCTGACCCGTGGCTTTACCGAACTGGAAT TTGTGAACCCGTTTAAAGTGATTAACCGTAAAACCTATCTGAACTTTGATAAAGCG GTGTTTCGTATTAACATTGTGCCGGATGAAAACTATACCATTAACGAAGGCTTTA ACCTGGAAGGCGCGAACAGCAACGGCCAGAACACCGAAATTAACAGCCGTAACT TTACCCGTCTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTG CGTGGCATTATTCCGTTTAAAACCAAAAGCCTGGATGAAGGCTATAACAAAGCG CTGTAA

[0233] LC / A1 (A3 LHD)

[0234] Amino acid sequence (SEQ ID NO: 9): QB\650053.01105\92299987.1 Page 47 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0235] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRML LTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIH AGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSRDA YDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLTRG FTELEFVNPFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQN TEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL-

[0236] DNA sequence (SEQ ID NO: 10):

[0237] ATGCCGTTTGTGAACAAACAGTTTAACTATAAAGATCCGGTGAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCAGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGACCAAACTGTTTGAACGTATTTATAGCACCGATCTGGGCCGTATGC TGCTGACCAGCATTGTGCGTGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTCAGCCGGATGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGAAGTGCTGAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCAAATTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGGCCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGTTTAAAGTGAACACCAACGCGTATTATGAAA TGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGATACC AACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATAACCTGCAGAA CATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCACCACCCCGCTGCAGTATA TGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGAAGATGCGAGCGGCAAAATTAGCGTG AACAAAGCGGCGTTTAAAGAATTTTATCGTGTGCTGACCCGTGGCTTTACCGAACTGGAATT TGTGAACCCGTTTAAAGTGCTGAACAGAAAAACCTATCTGAACTTTGATAAAGCGG TGTTTAAAATTAACATTGTGCCGAAAGTGAACTATACCATTTATGATGGCTTTAA CCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGCCAGAACACCGAAATTAA CAACATGAACTTTACCAAACTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAA QB\650053.01105\92299987.1 Page 48 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION CTGCTGTGTGTGCGTGGCATTATTACCAGCAAAACCAAAAGCCTGGATAAAGGC TATAACAAAGCGCTGTAA

[0238] LC / A3 (A1 LHD)

[0239] Amino acid sequence (SEQ ID NO: 11):

[0240] MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRML LSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSF GHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELI HAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRL YYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLT EIYTEDNFVKFFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEIN SRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0241] DNA sequence ((SEQ ID NO: 12):

[0242] ATGCCGTTTGTGAACAAACAATTTAACTATCGTGATCCGGTAAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCCATGATGC GAAATTTATTGATAGCCTCCAGGAAAACGAATTTCGTCTGTATTATTATAACAAATTTAAAG ATATTGCGAGCACCCTGAACAAAGCGAAAAGCATTGTGGGCACCACCGCGAGCCTCCAGTAT ATGAAAAACGTGTTTAAAGAAAAATATCTGCTGAGCGAAGATACCAGCGGCAAATTTAGCGT QB\650053.01105\92299987.1 Page 49 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION GGATAAACTGAAATTTGATAAACTGTATAAAATGCTGACCGAAATTTATACCGAAGATAACT TTGTGAAATTTTTTAAAGTGATTAACCGTAAAACCTATCTGAACTTTGATAAAGCG GTGTTTCGTATTAACATTGTGCCGGATGAAAACTATACCATTAACGAAGGCTTTA ACCTGGAAGGCGCGAACAGCAACGGCCAGAACACCGAAATTAACAGCCGTAACT TTACCCGTCTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTG CGTGGCATTATTCCGTTTAAAACCAAAAGCCTGGATGAAGGCTATAACAAAGCG CTGTAA

[0243] References for Examples 2 and 3

[0244] 1. Heap, J.T.; Pennington, O.J.; Cartman, S.T.; Minton, N.P. A modular 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].

[0245] 2. Bradshaw, M.; Tepp, W.H.; Whitemarsh, R.C.; Pellett, S.; Johnson, E.A. Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System. Appl Environ Microbiol 2014, 80, 7415-7422, doi:10.1128 / aem.01795-14.

[0246] 3. Malizio, C.J.; Goodnough, M.C.; Johnson, E.A. Purification of Clostridium botulinum type A neurotoxin. Methods in molecular biology (Clifton, N.J.) 2000, 145, 27-39, doi:10.1385 / 1-59259-052-7:27.

[0247] 4. Moberg, L.J.; Sugiyama, H. Affinity chromatography purification of type A botulinum neurotoxin from crystalline toxic complex. Appl Environ Microbiol 1978, 35, 878- 880.

[0248] 5. Hatheway, C.L. Botulism. In Laboratory diagnosis of infectious diseases: principles and practice., Balows, A., Hausler, W.H., Ohashi, M., Turano, M.A., Eds. Springer- Verlag: New York, 1988; Vol.1, pp.111–133.

[0249] 6. Schantz, E.J.; Kautter, D.A. Standardized assay for Clostridium botulinum toxins. Journal - Association of Official Analytical Chemists 1978, 61, 96-99.

[0250] 7. Reed, L.; Muench, H. A simple method of estimating fifty percent endpoints. Am. J. Hyg.1938, 27, 493-497.

[0251] 8. Pellett, S.; Tepp, W.H.; Clancy, C.M.; Borodic, G.E.; Johnson, E.A. A neuronal cell-based botulinum neurotoxin assay for highly sensitive and specific detection of neutralizing serum antibodies. FEBS letters 2007, 581, 4803-4808, doi:10.1016 / j.febslet.2007.08.078. QB\650053.01105\92299987.1 Page 50 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0252] 9. Pellett, S.; Tepp, W.H.; Toth, S.I.; Johnson, E.A. Comparison of the primary rat spinal cord cell (RSC) assay and the mouse bioassay for botulinum neurotoxin type A potency determination. Journal of pharmacological and toxicological methods 2010, 61, 304-310, doi:10.1016 / j.vascn.2010.01.003.

[0253] 10. Tepp, W.H.; Lin, G.; Johnson, E.A. Purification and characterization of a novel subtype a3 botulinum neurotoxin. Appl Environ Microbiol 2012, 78, 3108-3113, doi:10.1128 / aem.07967-11.

[0254] 11. Gardner, A.; Tepp, W.H.; Bradshaw, M.; Barbieri, J.T.; Pellett, S. Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane. International journal of molecular sciences 2021, 22, doi:10.3390 / ijms222011115.

[0255] 12. Whitemarsh, R.C.; Strathman, M.J.; Chase, L.G.; Stankewicz, C.; Tepp, W.H.; Johnson, E.A.; Pellett, S. Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection. Toxicological sciences : an official journal of the Society of Toxicology 2012, 126, 426-435, doi:10.1093 / toxsci / kfr354.

[0256] Additional Sequences – mRNA Sequences

[0257] >WT LC / A1 (SEQ ID NO: 39)

[0258] ATGCCGTTTGTGAACAAACAGTTTAACTATAAAGATCCGGTGAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCAGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGACCAAACTGTTTGAACGTATTTATAGCACCGATCTGGGCCGTATGC TGCTGACCAGCATTGTGCGTGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTCAGCCGGATGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGAAGTGCTGAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCAAATTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGGCCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGTTTAAAGTGAACACCAACGCGTATTATGAAA TGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCCATGATG CGAAATTTATTGATAGCCTCCAGGAAAACGAATTTCGTCTGTATTATTATAACAA ATTTAAAGATATTGCGAGCACCCTGAACAAAGCGAAAAGCATTGTGGGCACCAC QB\650053.01105\92299987.1 Page 51 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION CGCGAGCCTCCAGTATATGAAAAACGTGTTTAAAGAAAAATATCTGCTGAGCGA AGATACCAGCGGCAAATTTAGCGTGGATAAACTGAAATTTGATAAACTGTATAA AATGCTGACCGAAATTTATACCGAAGATAACTTTGTGAAATTTTTTAAAGTGCTG AACAGAAAAACCTATCTGAACTTTGATAAAGCGGTGTTTAAAATTAACATTGTGC CGAAAGTGAACTATACCATTTATGATGGCTTTAACCTGCGTAACACCAACCTGGC GGCGAACTTTAACGGCCAGAACACCGAAATTAACAACATGAACTTTACCAAACT GAAAAACTTTACCGGCCTGTTTGAATTTTATAAACTGCTGTGTGTGCGTGGCATT ATTACCAGCAAAACCAAAAGCCTGGATAAAGGCTATAACAAAGCGCTGTAA

[0259] >WT LC / A3 (SEQ ID NO: 40)

[0260] ATGCCGTTTGTGAACAAACAATTTAACTATCGTGATCCGGTAAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGAT ACCAACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATA ACCTGCAGAACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCA CCACCCCGCTGCAGTATATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGA AGATGCGAGCGGCAAAATTAGCGTGAACAAAGCGGCGTTTAAAGAATTTTATCG TGTGCTGACCCGTGGCTTTACCGAACTGGAATTTGTGAACCCGTTTAAAGTGATT AACCGTAAAACCTATCTGAACTTTGATAAAGCGGTGTTTCGTATTAACATTGTGC CGGATGAAAACTATACCATTAACGAAGGCTTTAACCTGGAAGGCGCGAACAGCA ACGGCCAGAACACCGAAATTAACAGCCGTAACTTTACCCGTCTGAAAAACTTTA CCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTGCGTGGCATTATTCCGTTTAAA ACCAAAAGCCTGGATGAAGGCTATAACAAAGCGCTGTAA QB\650053.01105\92299987.1 Page 52 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0261] >JV LC / A3 (SEQ ID NO: 41)

[0262] ATGCCGTTTGTGAACAAACCGTTTAACTATCGTGATCCGGGCAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGAT ACCAACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATA ACCTGCAGAACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCA CCACCCCGCTGCAGTATATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGA AGATGCGAGCGGCAAAATTAGCGTGAACAAAGCGGCGTTTAAAGAATTTTATCG TGTGCTGACCCGTGGCTTTACCGAACTGGAATTTGTGAACCCGTTTAAAGTGATT AACCGTAAAACCTATCTGAACTTTGATAAAGCGGTGTTTCGTATTAACATTGTGC CGGATGAAAACTATACCATTAACGAAGGCTTTAACCTGGAAGGCGCGAACAGCA ACGGCCAGAACACCGAAATTAACAGCCGTAACTTTACCCGTCTGAAAAACTTTA CCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTGCGTGGCATTATTCCGTTTAAA ACCAAAAGCCTGGATGAAGGCTATAACAAAGCGCTGTAA

[0263] >LC / A1 (A3 LHD) (SEQ ID NO: 42)

[0264] ATGCCGTTTGTGAACAAACAGTTTAACTATAAAGATCCGGTGAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCAGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGACCAAACTGTTTGAACGTATTTATAGCACCGATCTGGGCCGTATGC QB\650053.01105\92299987.1 Page 53 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION TGCTGACCAGCATTGTGCGTGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTCAGCCGGATGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGAAGTGCTGAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCAAATTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGGCCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGTTTAAAGTGAACACCAACGCGTATTATGAAA TGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGATA CCAACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATAA CCTGCAGAACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCAC CACCCCGCTGCAGTATATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGAA GATGCGAGCGGCAAAATTAGCGTGAACAAAGCGGCGTTTAAAGAATTTTATCGT GTGCTGACCCGTGGCTTTACCGAACTGGAATTTGTGAACCCGTTTAAAGTGCTGA ACAGAAAAACCTATCTGAACTTTGATAAAGCGGTGTTTAAAATTAACATTGTGCC GAAAGTGAACTATACCATTTATGATGGCTTTAACCTGCGTAACACCAACCTGGCG GCGAACTTTAACGGCCAGAACACCGAAATTAACAACATGAACTTTACCAAACTG AAAAACTTTACCGGCCTGTTTGAATTTTATAAACTGCTGTGTGTGCGTGGCATTAT TACCAGCAAAACCAAAAGCCTGGATAAAGGCTATAACAAAGCGCTGTAA

[0265] >LC / A3 (A1 LHD) (SEQ ID NO: 43)

[0266] ATGCCGTTTGTGAACAAACAATTTAACTATCGTGATCCGGTAAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC QB\650053.01105\92299987.1 Page 54 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCCATGAT GCGAAATTTATTGATAGCCTCCAGGAAAACGAATTTCGTCTGTATTATTATAACA AATTTAAAGATATTGCGAGCACCCTGAACAAAGCGAAAAGCATTGTGGGCACCA CCGCGAGCCTCCAGTATATGAAAAACGTGTTTAAAGAAAAATATCTGCTGAGCG AAGATACCAGCGGCAAATTTAGCGTGGATAAACTGAAATTTGATAAACTGTATA AAATGCTGACCGAAATTTATACCGAAGATAACTTTGTGAAATTTTTTAAAGTGAT TAACCGTAAAACCTATCTGAACTTTGATAAAGCGGTGTTTCGTATTAACATTGTG CCGGATGAAAACTATACCATTAACGAAGGCTTTAACCTGGAAGGCGCGAACAGC AACGGCCAGAACACCGAAATTAACAGCCGTAACTTTACCCGTCTGAAAAACTTT ACCGGCCTGTTTGAATTTTATAAACTGCTGTGCGTGCGTGGCATTATTCCGTTTAA AACCAAAAGCCTGGATGAAGGCTATAACAAAGCGCTGTAA

[0267] Example 4

[0268] As our mRNA study indicated the N-terminus of the BoNT / A LC can affect duration of action in addition to the LHD, and we had identified the MLD, we further investigated the effect of the MLD and the N-terminus (A3 -Q7P, V14G) in our hiPSC derived motor-neurons-mRNA assay. Since the nanoparticles from Ribopro had provided better transformation efficacy, the experiment was conducted using these nanoparticles for mRNA delivery.

[0269] Methods

[0270] Neuronal Cell Culture. Spinal motor neurons derived from human induced pluripotent stem cells (hiPSCs) were provided by BrainXell (Madison, WI) and used essentially as previously described

[0012] . The cells were seeded into 96-well TPP plates (Techno Plastic Products, Midwest Scientific, Valley Park, MO) that had been coated with 0.01% poly-L- ornithine and 8.3 µg / cm2 Matrigel (BD Biosciences, East Rutherford, NJ) at a density of about 38,000 cells per well and maintained as per manufacturer’s instructions. Recommended culture media was used, comprised of equal parts Neurobasal and DMEM / F12 Medium supplemented with B27, N2 and GlutaMAX (Life Technologies). Cells were cultured for 3 days prior to transfection with mRNA and for 7 days prior transfection with LC / A1 mRNA in LNP formulation.

[0271] mRNA Constructs. All mRNA constructs were purchased from RiboPro (Netherlands) and were produced with the following modifications: ^ RNA synthesis from de novo QB\650053.01105\92299987.1 Page 55 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION ^ template / plasmid ^ dsRNA reduced ^ Cap1 upgrade ^ 150nt Poly-A tail ^ Buffer: RNAse-Free-Water ^ Concentration: 1 μg / μl For the lipid formulation BoNT / A1 LC mRNA, production was as follows: ^ RNA synthesis from de novo ^ template / plasmid ^ dsRNA reduced ^ Cap1 upgrade ^ 150nt Poly-A tail ^ Formulated in ciLNP in PBS ^ Concentration: 100 ng / μl

[0272] The following constructs were created: LC / A1 aa1-450, LC / A3 aa 1-446, 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 sequences and nucleotide sequences are provided below.

[0273] RiboPro LNP formulation Transfection. 7 Days after seeding hiPSC-derived motor neurons, the cells were transfected with LC / A1 mRNA in RiboPro’s lipid nanoparticle formulation. 100 or 200 ng LC / A1 mRNA in LNP per well was added directly to the cells or was first diluted in OptiMEM media before adding to cells. Cells were incubated with complexes at 37 ^C and 5% CO2 and samples were collected in 1 x 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 store at -20 ^C for Western Blot analysis. Note: the LC / A1 mRNA formulated in nanoparticles had expired, and thus this mRNA was transfected using RNAMax transfection reagent as described above.

[0274] Western Blot Analysis. Cell lysates were analyzed by Western blot and densitometry for the percentage of SNAP-25 that had been cleaved using a monoclonal anti- SNAP-25 antibody that recognizes both cleaved and uncleaved SNAP-25 (Synaptic Systems, Germany) as previously described ([8,9]). Images were obtained using PhosphaGlo reagent (KPL, Gaithersburg, MD) and an Azure C600 Imaging system. Densitometry was conducted using the Azure spot software version 2.0.062, and average and standard deviations of the five QB\650053.01105\92299987.1 Page 56 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION replicates were used to create a graph of cleaved SNAP-25 (in percent compared to un-cleaved) versus time in excel. The data points were fitted with logarithmic trendlines.

[0275] Results

[0276] BoNT / A LC mRNA transfection of neuronal cells results in functional intra- neuronal LC in all to almost all cells, with duration of action determined by N-terminus and MLD. Transfection of the motor-neurons with a GFP control mRNA showed nearly 100% of cells had been transfected. For the BoNT / A LC constructs, transfection efficiency was evaluated by percent cleaved SNAP-25 at the first time point 6 days post transfection. At this time point, percent SNAP-25 cleavage ranged from 77 % to 100%, with the exception of BoNT / A1 LC, which had been transfected using RNA Max reagent and resulted in 37% SNAP- 25 cleavage. (Fig. 14). The lower values of some of the constructs correlated to lower LC potency previously determined for holotoxins (see Example 2). This indicates excellent transfection efficiency sufficient for therapeutic utility. Decrease of the percent cleaved SNAP- 25 was monitored up to 56 days post transfection.

[0277] For all LC constructs except LC / A1, a logarithmic decrease in percent cleaved SNAP-25 was observed. LC / A1-treated cells maintained constant levels of cleaved SNAP-25 over the tested time period. The most rapid decrease in cleaved SNAP-25 was observed for the LC / A3-Q7P, V14G, the LC / A3-Q7P, V14G-A1MLD, and LC / A1-A3MLD. This indicates that the Q7P, V14G mutations in the LC / A N-terminus are dominant in determining a short duration of action, and that the A3-MLD determines a similarly short duration of action. The LC / A3wt and LC / A3wt-A1MLD resulted in a similar and slower decrease in cleaved SNAP- 25. This indicates that the A1MLD is not sufficient to establish longer duration of action in the LC / A3, in spite of its ability to direct membrane localization of 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). 2022 Nov 22;14(12):814. doi: 10.3390 / toxins14120814. PMID: 36548711; PMCID: PMC9783275). Overall, the data is consistent with the mRNA data of the LHD mutant, with the exception that the A1-MLD did not prolong duration of action, whereas the A1-LHD appeared to potentially slightly increase duration of action of LC / A3 (FIG.11).

[0278] Discussion

[0279] These data demonstrate highly efficient transfection of human neurons with BoNT / A LC mRNA, resulting in production of functionally active LC inside the targeted neurons. LC activity persisted for a prolonged time consistent with activity observed in hiPSC derived neurons intoxicated with BoNT / A protein. The data further show that both the N- QB\650053.01105\92299987.1 Page 57 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION terminus and the MLD play a role the in regulating duration of action of BoNT / A LC. Taken together these data indicate feasibility of utilizing BoNT LC mRNA constructs therapeutically as an alternative to protein based therapy. Further, the duration of action can be customized through sequence modification of the mRNA in the N-terminus and in the MLD domain. Since nanoparticle targeting is not restricted to neurons, additional targets including inflammatory cells or cancer cells are feasible targets for such a therapy. Additionally, neuronal sub- population such as pain-transmitting neurons, may be targeted with this technology.

[0280] Based upon the data in FIG. 14, both the A1-N-Term and A1-MLD contribute to the long duration of BoNT / A1 action. These data support a role for both LC / A1 N terminus (N-Term) and MLD, in addition to the LHD, in eliciting a Two Step mechanism where the N terminus (A1-N-Term residues 1-17 of A1) mediates Rab-dependent vesicle trafficking of LC / A1 to the plasma membrane, where the A1-MLD (residues 275-334 of A1) mediates stable association with the plasma membrane; these two steps elicit the high potency and long duration of BoNT / A1 action. Based upon 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 BoNT potency and duration of action.

[0281] Sequences for Example 4

[0282] >WT LC / A1 (SEQ ID NO: 44)

[0283] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRML LTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIH AGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLY YYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKML TEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFN GQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL-

[0284] DNA seq* (SEQ ID NO: 45)

[0285] ATGCCGTTTGTGAACAAACAGTTTAACTATAAAGATCCGGTGAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCAGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGACCAAACTGTTTGAACGTATTTATAGCACCGATCTGGGCCGTATGC QB\650053.01105\92299987.1 Page 58 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION TGCTGACCAGCATTGTGCGTGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTCAGCCGGATGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGAAGTGCTGAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCAAATTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGGCCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGTTTAAAGTGAACACCAACGCGTATTATGAAA TGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCCATGATG CGAAATTTATTGATAGCCTCCAGGAAAACGAATTTCGTCTGTATTATTATAACAAATTTAA AGATATTGCGAGCACCCTGAACAAAGCGAAAAGCATTGTGGGCACCACCGCGAGCCTCCAGT ATATGAAAAACGTGTTTAAAGAAAAATATCTGCTGAGCGAAGATACCAGCGGCAAATTTAGC GTGGATAAACTGAAATTTGATAAACTGTATAAAATGCTGACCGAAATTTATACCG AAGATAACTTTGTGAAATTTTTTAAAGTGCTGAACAGAAAAACCTATCTGAACTT TGATAAAGCGGTGTTTAAAATTAACATTGTGCCGAAAGTGAACTATACCATTTAT GATGGCTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGCCAGAAC ACCGAAATTAACAACATGAACTTTACCAAACTGAAAAACTTTACCGGCCTGTTTG AATTTTATAAACTGCTGTGTGTGCGTGGCATTATTACCAGCAAAACCAAAAGCCT GGATAAAGGCTATAACAAAGCGCTGTAA

[0286] >WT LC / A1 (A3 MLD) (SEQ ID NO: 46)

[0287] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRML LTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIH AGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLWQKKFSRD AYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKLKFDKLYKML TEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFN GQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL-

[0288] DNA seq* (SEQ ID NO: 47)

[0289] ATGCCGTTTGTGAACAAACAGTTTAACTATAAAGATCCGGTGAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCAGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG QB\650053.01105\92299987.1 Page 59 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGACCAAACTGTTTGAACGTATTTATAGCACCGATCTGGGCCGTATGC TGCTGACCAGCATTGTGCGTGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTCAGCCGGATGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTATTGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGAAGTGCTGAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCAAATTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGGCCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGTTTAAAGTGAACACCAACGCGTATTATGAAA TGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCCATGATG CGAAATTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATAACCTGCA GAACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCACCACCCCGCTGCAGT ATATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGAAGATGCGAGCGGCAAAATTAGC GTGAACAAACTGAAATTTGATAAACTGTATAAAATGCTGACCGAAATTTATACCG AAGATAACTTTGTGAAATTTTTTAAAGTGCTGAACAGAAAAACCTATCTGAACTT TGATAAAGCGGTGTTTAAAATTAACATTGTGCCGAAAGTGAACTATACCATTTAT GATGGCTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGCCAGAAC ACCGAAATTAACAACATGAACTTTACCAAACTGAAAAACTTTACCGGCCTGTTTG AATTTTATAAACTGCTGTGTGTGCGTGGCATTATTACCAGCAAAACCAAAAGCCT GGATAAAGGCTATAACAAAGCGCTGTAA

[0290] >JV LC / A3 (SEQ ID NO: 48)

[0291] MPFVNKPFNYRDPGNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRML LSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSF GHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELI HAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSR DAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVL TRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEI NSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0292] DNA seq* (SEQ ID NO: 49)

[0293] ATGCCGTTTGTGAACAAACCGTTTAACTATCGTGATCCGGGCAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA QB\650053.01105\92299987.1 Page 60 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGAT ACCAACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATAACCTGC AGAACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCACCACCCCGCTGCAG TATATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGAAGATGCGAGCGGCAAAATTAG CGTGAACAAAGCGGCGTTTAAAGAATTTTATCGTGTGCTGACCCGTGGCTTTACCG AACTGGAATTTGTGAACCCGTTTAAAGTGATTAACCGTAAAACCTATCTGAACTT TGATAAAGCGGTGTTTCGTATTAACATTGTGCCGGATGAAAACTATACCATTAAC GAAGGCTTTAACCTGGAAGGCGCGAACAGCAACGGCCAGAACACCGAAATTAAC AGCCGTAACTTTACCCGTCTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAAC TGCTGTGCGTGCGTGGCATTATTCCGTTTAAAACCAAAAGCCTGGATGAAGGCTA TAACAAAGCGCTGTAA

[0294] >JV LC / A3 (A1 MLD) (SEQ ID NO: 50)

[0295] MPFVNKPFNYRDPGNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRML LSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSF GHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELI HAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLQENEFRL YYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKAAFKEFYRVL TRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEI NSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0296] DNA Seq* (SEQ ID NO: 51) QB\650053.01105\92299987.1 Page 61 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0297] ATGCCGTTTGTGAACAAACCGTTTAACTATCGTGATCCGGGCAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGAT ACCAACTTTATTGATAGCCTCCAGGAAAACGAATTTCGTCTGTATTATTATAACAAATTTA AAGATATTGCGAGCACCCTGAACAAAGCGAAAAGCATTGTGGGCACCACCGCGAGCCTCCAG TATATGAAAAACGTGTTTAAAGAAAAATATCTGCTGAGCGAAGATACCAGCGGCAAATTTAG CGTGGATAAAGCGGCGTTTAAAGAATTTTATCGTGTGCTGACCCGTGGCTTTACCG AACTGGAATTTGTGAACCCGTTTAAAGTGATTAACCGTAAAACCTATCTGAACTT TGATAAAGCGGTGTTTCGTATTAACATTGTGCCGGATGAAAACTATACCATTAAC GAAGGCTTTAACCTGGAAGGCGCGAACAGCAACGGCCAGAACACCGAAATTAAC AGCCGTAACTTTACCCGTCTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAAC TGCTGTGCGTGCGTGGCATTATTCCGTTTAAAACCAAAAGCCTGGATGAAGGCTA TAACAAAGCGCTGTAA

[0298] >LM LC / A3 (SEQ ID NO: 52)

[0299] MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRML LSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSF GHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELI HAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSR DAYDNLQNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVL QB\650053.01105\92299987.1 Page 62 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION TRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEI NSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0300] DNA Seq* (SEQ ID NO: 53)

[0301] ATGCCGTTTGTGAACAAACAATTTAACTATCGTGATCCGGTAAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGAT ACCAACTTTATTGATAGCCTGTGGCAGAAAAAATTTAGCCGTGATGCGTATGATAACCTGC AGAACATTGCGCGTATTCTGAACGAAGCGAAAACCATTGTGGGCACCACCACCCCGCTGCAG TATATGAAAAACATTTTTATTCGTAAATATTTTCTGAGCGAAGATGCGAGCGGCAAAATTAG CGTGAACAAAGCGGCGTTTAAAGAATTTTATCGTGTGCTGACCCGTGGCTTTACCG AACTGGAATTTGTGAACCCGTTTAAAGTGATTAACCGTAAAACCTATCTGAACTT TGATAAAGCGGTGTTTCGTATTAACATTGTGCCGGATGAAAACTATACCATTAAC GAAGGCTTTAACCTGGAAGGCGCGAACAGCAACGGCCAGAACACCGAAATTAAC AGCCGTAACTTTACCCGTCTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAAC TGCTGTGCGTGCGTGGCATTATTCCGTTTAAAACCAAAAGCCTGGATGAAGGCTA TAACAAAGCGCTGTAA

[0302] >LM LC / A3 (A1MLD) (SEQ ID NO: 54)

[0303] MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRML LSFIVKGIPFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSF GHDVFNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELI QB\650053.01105\92299987.1 Page 63 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION HAAHRLYGIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLQENEFRL YYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKAAFKEFYRVL TRGFTELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEI NSRNFTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKAL-

[0304] DNA Seq* (SEQ ID NO: 55)

[0305] ATGCCGTTTGTGAACAAACAATTTAACTATCGTGATCCGGTAAACGGC GTGGATATTGCGTATATTAAAATTCCGAACGCGGGCCAGATGCAGCCGGTGAAA GCGTTTAAAATTCATGAAGGCGTGTGGGTGATTCCGGAACGTGATACCTTTACCA ACCCGGAAGAAGGCGATCTGAACCCGCCGCCGGAAGCGAAACAGGTGCCGGTG AGCTATTATGATAGCACCTATCTGAGCACCGATAACGAAAAAGATAACTATCTG AAAGGCGTGATTAAACTGTTTGATCGTATTTATAGCACCGGCCTGGGCCGTATGC TGCTGAGCTTTATTGTGAAAGGCATTCCGTTTTGGGGCGGCAGCACCATTGATAC CGAACTGAAAGTGATTGATACCAACTGCATTAACGTGATTGAACCGGGCGGCAG CTATCGTAGCGAAGAACTGAACCTGGTGATTACCGGCCCGAGCGCGGATATTATT CAGTTTGAATGCAAAAGCTTTGGCCATGATGTGTTTAACCTGACCCGTAACGGCT ATGGCAGCACCCAGTATATTCGTTTTAGCCCGGATTTTACCTTTGGCTTTGAAGA AAGCCTGGAAGTGGATACCAACCCGCTGCTGGGCGCGGGCACCTTTGCGACCGA TCCGGCGGTGACCCTGGCGCATGAACTGATTCATGCGGCGCATCGTCTGTATGGC ATTGCGATTAACCCGAACCGTGTGCTGAAAGTGAAAACCAACGCGTATTATGAA ATGAGCGGCCTGGAAGTGAGCTTTGAAGAACTGCGTACCTTTGGCGGCAACGAT ACCAACTTTATTGATAGCCTCCAGGAAAACGAATTTCGTCTGTATTATTATAACAAATTTA AAGATATTGCGAGCACCCTGAACAAAGCGAAAAGCATTGTGGGCACCACCGCGAGCCTCCAG TATATGAAAAACGTGTTTAAAGAAAAATATCTGCTGAGCGAAGATACCAGCGGCAAATTTAG CGTGGATAAAGCGGCGTTTAAAGAATTTTATCGTGTGCTGACCCGTGGCTTTACCG AACTGGAATTTGTGAACCCGTTTAAAGTGATTAACCGTAAAACCTATCTGAACTT TGATAAAGCGGTGTTTCGTATTAACATTGTGCCGGATGAAAACTATACCATTAAC GAAGGCTTTAACCTGGAAGGCGCGAACAGCAACGGCCAGAACACCGAAATTAAC AGCCGTAACTTTACCCGTCTGAAAAACTTTACCGGCCTGTTTGAATTTTATAAAC TGCTGTGCGTGCGTGGCATTATTCCGTTTAAAACCAAAAGCCTGGATGAAGGCTA TAACAAAGCGCTGTAA

[0306] Further Sequences 1. BoNT / E QB\650053.01105\92299987.1 Page 64 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION a. Amino acid sequence LC / E 1-426 (SEQ ID NO: 13) 1 MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS 61 LKNGDSSYYD PNYLQSDEEK DRFLKIVTKI FNRINNNLSG GILLEELSKA NPYLGNDNTP 121 DNQFHIGDAS AVEIKFSNGS QDILLPNVII MGAEPDLFET NSSNISLRNN YMPSNHGFGS 181 IAIVTFSPEY SFRFNDNSMN EFIQDPALTL MHELIHSLHG LYGAKGITTK YTITQKQNPL 241 ITNIRGTNIE EFLTFGGTDL NIITSAQSND IYTNLLADYK KIASKLSKVQ VSNPLLNPYK 301 DVFEAKYGLD KDASGIYSVN INKFNDIFKK LYSFTEFDLA TKFQVKCRQT YIGQYKYFKL 361 SNLLNDSIYN ISEGYNINNL KVNFRGQNAN LNPRIITPIT GRGLVKKIIR FC a. Amino acid sequence BoNT / E 1-1251 (SEQ ID NO: 14) 1 MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS 61 LKNGDSSYYD PNYLQSDEEK DRFLKIVTKI FNRINNNLSG GILLEELSKA NPYLGNDNTP 121 DNQFHIGDAS AVEIKFSNGS QDILLPNVII MGAEPDLFET NSSNISLRNN YMPSNHRFGS 181 IAIVTFSPEY SFRFNDNCMN EFIQDPALTL MHELIHSLHG LYGAKGITTK YTITQKQNPL 241 ITNIRGTNIE EFLTFGGTDL NIITSAQSND IYTNLLADYK KIASKLSKVQ VSNPLLNPYK 301 DVFEAKYGLD KDASGIYSVN INKFNDIFKK LYSFTEFDLR TKFQVKCRQT YIGQYKYFKL 361 SNLLNDSIYN ISEGYNINNL KVNFRGQNAN LNPRIITPIT GRGLVKKIIR FCKNIVSVKG 421 IRKSICIEIN NGELFFVASE NSYNDDNINT PKEIDDTVTS NNNYENDLDQ VILNFNSESA 481 PGLSDEKLNL TIQNDAYIPK YDSNGTSDIE QHDVNELNVF FYLDAQKVPE GENNVNLTSS 541 IDTALLEQPK IYTFFSSEFI NNVNKPVQAA LFVSWIQQVL VDFTTEANQK STVDKIADIS 601 IVVPYIGLAL NIGNEAQKGN FKDALELLGA GILLEFEPEL LIPTILVFTI KSFLGSSDNK 661 NKVIKAINNA LKERDEKWKE VYSFIVSNWM TKINTQFNKR KEQMYQALQN QVNAIKTIIE 721 SKYNSYTLEE KNELTNKYDI KQIENELNQK VSIAMNNIDR FLTESSISYL MKIINEVKIN 781 KLREYDENVK TYLLNYIIQH GSILGESQQE LNSMVTDTLN NSIPFKLSSY TDDKILISYF 841 NKFFKRIKSS SVLNMRYKND KYVDTSGYDS NININGDVYK YPTNKNQFGI YNDKLSEVNI 901 SQNDYIIYDN KYKNFSISFW VRIPNYDNKI VNVNNEYTII NCMRDNNSGW KVSLNHNEII 961 WTFEDNRGIN QKLAFNYGNA NGISDYINKW IFVTITNDRL GDSKLYINGN LIDQKSILNL 1021 GNIHVSDNIL FKIVNCSYTR YIGIRYFNIF DKELDETEIQ TLYSNEPNTN ILKDFWGNYL 1081 LYDKEYYLLN VLKPNNFIDR RKDSTLSINN IRSTILLANR LYSGIKVKIQ RVNNSSTNDN 1141 LVRKNDQVYI NFVASKTHLF PLYADTATTN KEKTIKISSS GNRFNQVVVM NSVGNCTMNF 1201 KNNNGNNIGL LGFKADTVVA STWYYTHMRD HTNSNGCFWN FISEEHGWQE K b. DNA sequence BoNT / E (SEQ ID NO: 15) 1 atgccaaaaa ttaatagttt taattataat gatcctgtta atgatagaac aattttatat 61 attaaaccag gcggttgtca agaattttat aaatcattta atattatgaa aaatatttgg 121 ataattccag agagaaatgt aattggtaca accccccaag attttcatcc gcctacttca 181 ttaaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtga tgaagaaaag 241 gatagatttt taaaaatagt cacaaaaata tttaatagaa taaataataa tctttcagga 301 gggattttat tagaagaact gtcaaaagct aatccatatt tagggaatga taatactcca 361 gataatcaat tccatattgg tgatgcatca gcagttgaga ttaaattctc aaatggtagc 421 caagacatac tattacctaa tgttattata atgggagcag agcctgattt atttgaaact 481 aacagttcca atatttctct aagaaataat tatatgccaa gcaatcaccg ttttggatca 541 atagctatag taacattctc acctgaatat tcttttagat ttaatgataa ttgtatgaat 601 gaatttattc aagatcctgc tcttacatta atgcatgaat taatacattc attacatgga 661 ctatatgggg ctaaagggat tactacaaag tatactataa cacaaaaaca aaatccccta 721 ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggagg tactgattta 781 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc tgattataaa QB\650053.01105\92299987.1 Page 65 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 841 aaaatagcgt ctaaacttag caaagtacaa gtatctaatc cactacttaa tccttataaa 901 gatgtttttg aagcaaagta tggattagat aaagatgcta gcggaattta ttcggtaaat 961 ataaacaaat ttaatgatat ttttaaaaaa ttatacagct ttacggaatt tgatttacga 1021 actaaatttc aagttaaatg taggcaaact tatattggac agtataaata cttcaaactt 1081 tcaaacttgt taaatgattc tatttataat atatcagaag gctataatat aaataattta 1141 aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta gaattattac accaattaca 1201 ggtagaggac tagtaaaaaa aatcattaga ttttgtaaaa atattgtttc tgtaaaaggc 1261 ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgt ggcttccgag 1321 aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatac agtaacttca 1381 aataataatt atgaaaatga tttagatcag gttattttaa attttaatag tgaatcagca 1441 cctggacttt cagatgaaaa attaaattta actatccaaa atgatgctta tataccaaaa 1501 tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaact taatgtattt 1561 ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatct cacctcttca 1621 attgatacag cattattaga acaacctaaa atatatacat ttttttcatc agaatttatt 1681 aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggataca acaagtgtta 1741 gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgc agatatttct 1801 atagttgttc catatatagg tcttgcttta aatataggaa atgaagcaca aaaaggaaat 1861 tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttga acccgagctt 1921 ttaattccta caattttagt attcacgata aaatcttttt taggttcatc tgataataaa 1981 aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaa atggaaagaa 2041 gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatt taataaaaga 2101 aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg caattaaaac aataatagaa 2161 tctaagtata atagttatac tttagaggaa aaaaatgagc ttacaaataa atatgatatt 2221 aagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataa tatagacagg 2281 ttcttaactg aaagttctat atcctattta atgaaaataa taaatgaagt aaaaattaat 2341 aaattaagag aatatgatga gaatgtcaaa acgtatttat tgaattatat tatacaacat 2401 ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaactga taccctaaat 2461 aatagtattc cttttaagct ttcttcttat acagatgata aaattttaat ttcatatttt 2521 aataaattct ttaagagaat taaaagtagt tcagttttaa atatgagata taaaaatgat 2581 aaatacgtag atacttcagg atatgattca aatataaata ttaatggaga tgtatataaa 2641 tatccaacta ataaaaatca atttggaata tataatgata aacttagtga agttaatata 2701 tctcaaaatg attacattat atatgataat aaatataaaa attttagtat tagtttttgg 2761 gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaata cactataata 2821 aattgtatga gagataataa ttcaggatgg aaagtatctc ttaatcataa tgaaataatt 2881 tggacattcg aagataatcg aggaattaat caaaaattag catttaacta tggtaacgca 2941 aatggtattt ctgattatat aaataagtgg atttttgtaa ctataactaa tgatagatta 3001 ggagattcta aactttatat taatggaaat ttaatagatc aaaaatcaat tttaaattta 3061 ggtaatattc atgttagtga caatatatta tttaaaatag ttaattgtag ttatacaaga 3121 tatattggta ttagatattt taatattttt gataaagaat tagatgaaac agaaattcaa 3181 actttatata gcaatgaacc taatacaaat attttgaagg atttttgggg aaattatttg 3241 ctttatgaca aagaatacta tttattaaat gtgttaaaac caaataactt tattgatagg 3301 agaaaagatt ctactttaag cattaataat ataagaagca ctattctttt agctaataga 3361 ttatatagtg gaataaaagt taaaatacaa agagttaata atagtagtac taacgataat 3421 cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaac tcacttattt 3481 ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaat atcatcatct 3541 ggcaatagat ttaatcaagt agtagttatg aattcagtag gaaattgtac aatgaatttt 3601 aaaaataata atggaaataa tattgggttg ttaggtttca aggcagatac tgtcgttgct 3661 agtacttggt attatacaca tatgagagat catacaaaca gcaatggatg tttttggaac 3721 tttatttctg aagaacatgg atggcaagaa aaataa c. LC / E coordinates from crystal structure PDB:1T3A and 3FFZ ^ N-Term 1-17 ^ MLD-R1264-287 ^ MLD-R2288-319 ^ MLD-R1-R2264-319 ^ LHD-R1-R2-R3264-343 ^ LC / E11-412 ^ BoNT / E 1251 QB\650053.01105\92299987.1 Page 66 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 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 DRLNKVLVCI SDPNININIY KNKFKDKYKF VEDSEGKYSI DVESFDKLYK SLMFGFTETN 361 IAENYKIKTR ASYFSDSLPP VKIKNLLDNE IYTIEEGFNI SDKDMEKEYR GQNKAINKQA 421 YEEISKEHLA VYKIQMC a. amino acid sequence BoNT / B 1-1291 (SEQ ID NO: 17) 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 DRLNKVLVCI SDPNININIY KNKFKDKYKF VEDSEGKYSI DVESFDKLYK SLMFGFTETN 361 IAENYKIKTR ASYFSDSLPP VKIKNLLDNE IYTIEEGFNI SDKDMEKEYR GQNKAINKQA 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 IFNTELSQSN IEERYKIQSY 1081 SEYLKDFWGN PLMYNKEYYM FNAGNKNSYI KLKKDSPVGE ILTRSKYNQN SKYINYRDLY 1141 IGEKFIIRRK SNSQSINDDI VRKEDYIYLD FFNLNQEWRV YTYKYFKKEE EKLFLAPISD 1201 SDEFYNTIQI KEYDEQPTYS CQLLFKKDEE STDEIGLIGI HRFYESGIVF EEYKDYFCIS 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 QB\650053.01105\92299987.1 Page 67 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 541 tttgcatcaa gggaaggctt cgggggtata atgcaaatga agttttgccc agaatatgta 601 agcgtattta ataatgttca agaaaacaaa ggcgcaagta tatttaatag acgtggatat 661 ttttcagatc cagccttgat attaatgcat gaacttatac atgttttaca tggattatat 721 ggcattaaag tagatgattt accaattgta ccaaatgaaa aaaaattttt tatgcaatct 781 acagatgcta tacaggcaga agaactatat acatttggag gacaagatcc cagcatcata 841 actccttcta cggataaaag tatctatgat aaagttttgc aaaattttag agggatagtt 901 gatagactta acaaggtttt agtttgcata tcagatccta acattaatat taatatatat 961 aaaaataaat ttaaagataa atataaattc gttgaagatt ctgagggaaa atatagtata 1021 gatgtagaaa gttttgataa attatataaa agcttaatgt ttggttttac agaaactaat 1081 atagcagaaa attataaaat aaaaactaga gcttcttatt ttagtgattc cttaccacca 1141 gtaaaaataa aaaatttatt agataatgaa atctatacta tagaggaagg gtttaatata 1201 tctgataaag atatggaaaa agaatataga ggtcagaata aagctataaa taaacaagct 1261 tatgaagaaa ttagcaagga gcatttggct gtatataaga tacaaatgtg taaaagtgtt 1321 aaagctccag gaatatgtat tgatgttgat aatgaagatt tgttctttat agctgataaa 1381 aatagttttt cagatgattt atctaaaaac gaaagaatag aatataatac acagagtaat 1441 tatatagaaa atgacttccc tataaatgaa ttaattttag atactgattt aataagtaaa 1501 atagaattac caagtgaaaa tacagaatca cttactgatt ttaatgtaga tgttccagta 1561 tatgaaaaac aacccgctat aaaaaaaatt tttacagatg aaaataccat ctttcaatat 1621 ttatactctc agacatttcc tctagatata agagatataa gtttaacatc ttcatttgat 1681 gatgcattat tattttctaa caaagtttat tcattttttt ctatggatta tattaaaact 1741 gctaataaag tggtagaagc aggattattt gcaggttggg tgaaacagat agtaaatgat 1801 tttgtaatcg aagctaataa aagcaatact atggataaaa ttgcagatat atctctaatt 1861 gttccttata taggattagc tttaaatgta ggaaatgaaa cagctaaagg aaattttgaa 1921 aatgcttttg agattgcagg agccagtatt ctactagaat ttataccaga acttttaata 1981 cctgtagttg gagccttttt attagaatca tatattgaca ataaaaataa aattattaaa 2041 acaatagata atgctttaac taaaagaaat gaaaaatgga gtgatatgta cggattaata 2101 gtagcgcaat ggctctcaac agttaatact caattttata caataaaaga gggaatgtat 2161 aaggctttaa attatcaagc acaagcattg gaagaaataa taaaatacag atataatata 2221 tattctgaaa aagaaaagtc aaatattaac atcgatttta atgatataaa ttctaaactt 2281 aatgagggta ttaaccaagc tatagataat ataaataatt ttataaatgg atgttctgta 2341 tcatatttaa tgaaaaaaat gattccatta gctgtagaaa aattactaga ctttgataat 2401 gctctcaaaa aaaatttgtt aaattatata gatgaaaata aattatattt gattggaagt 2461 gcagaatatg aaaaatcaaa agtaaataaa tacttgaaaa ccattatgcc gtttgatctt 2521 tcaatatata ccaatgatac aatactaata gaaatgttta ataaatataa tagcgaaatt 2581 ttaaataata ttatcttaaa tttaagatat aaggataata atttaataga tttatcagga 2641 tatggggcaa aggtagaggt atatgatgga gtcgagctta atgataaaaa tcaatttaaa 2701 ttaactagtt cagcaaatag taagattaga gtgactcaaa atcagaatat catatttaat 2761 agtgtgttcc ttgattttag cgttagcttt tggataagaa tacctaaata taagaatgat 2821 ggtatacaaa attatattca taatgaatat acaataatta attgtatgaa aaataattcg 2881 ggctggaaaa tatctattag gggtaatagg ataatatgga ctttaattga tataaatgga 2941 aaaaccaaat cggtattttt tgaatataac ataagagaag atatatcaga gtatataaat 3001 agatggtttt ttgtaactat tactaataat ttgaataacg ctaaaattta tattaatggt 3061 aagctagaat caaatacaga tattaaagat ataagagaag ttattgctaa tggtgaaata 3121 atatttaaat tagatggtga tatagataga acacaattta tttggatgaa atatttcagt 3181 atttttaata cggaattaag tcaatcaaat attgaagaaa gatataaaat tcaatcatat 3241 agcgaatatt taaaagattt ttggggaaat cctttaatgt acaataaaga atattatatg 3301 tttaatgcgg ggaataaaaa ttcatatatt aaactaaaga aagattcacc tgtaggtgaa 3361 attttaacac gtagcaaata taatcaaaat tctaaatata taaattatag agatttatat 3421 attggagaaa aatttattat aagaagaaag tcaaattctc aatctataaa tgatgatata 3481 gttagaaaag aagattatat atatctagat ttttttaatt taaatcaaga gtggagagta 3541 tatacctata aatattttaa gaaagaggaa gaaaaattgt ttttagctcc tataagtgat 3601 tctgatgagt tttacaatac tatacaaata aaagaatatg atgaacagcc aacatatagt 3661 tgtcagttgc tttttaaaaa agatgaagaa agtactgatg agataggatt gattggtatt 3721 catcgtttct acgaatctgg aattgtattt gaagagtata aagattattt ttgtataagt 3781 aaatggtact taaaagaggt aaaaaggaaa ccatataatt taaaattggg atgtaattgg 3841 cagtttattc ctaaagatga agggtggact gaataa c.LC / B coordinates from protein structure PDB:1S0F add.Met ^ N-Term 1-17 ^ MLD-R1281-305 QB\650053.01105\92299987.1 Page 68 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION ^ MLD-R2306-340 ^ MLD-R1-R2280-340 ^ LHD-R1-R2-R3280-365 ^ 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 / C11-1291 (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 DINEETEVIY 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 LNPIFPFDFK LGSSGEDRGK VIVTQNENIV YNSMYESFSI SFWIRINKWV SNLPGYTIID 961 SVKNNSGWSI GIISNFLVFT LKQNEDSEQS INFSYDISNN APGYNKWFFV TVTNNMMGNM 1021 KIYINGKLID TIKVKELTGI NFSKTITFEI NKIPDTGLIT SDSDNINMWI RDFYIFAKEL 1081 DGKDINILFN SLQYTNVVKD YWGNDLRYNK EYYMVNIDYL NRYMYANSRQ IVFNTRRNNN 1141 DFNEGYKIII KRIRGNTNDT RVRGGDILYF DMTINNKAYN LFMKNETMYA DNHSTEDIYA 1201 IGLREQTKDI NDNIIFQIQP MNNTYYYASQ IFKSNFNGEN ISGICSIGTY RFRLGGDWYR 1261 HNYLVPTVKQ GNYASLLEST STHWGFVPVS E b. DNA sequence BoNT / C (SEQ ID NO: 21) QB\650053.01105\92299987.1 Page 69 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 1 atgccaataa caattaacaa ctttaattat tcagatcctg ttgataataa aaatatttta 61 tatttagata ctcatttaaa tacactagct aatgagcctg aaaaagcctt tcgcattacaQB\650053.01105\92299987.1 Page 70 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 3601 ataggtttaa gagaacaaac aaaggatata aatgataata ttatatttca aatacaacca 3661 atgaataata cttattatta cgcatctcaa atatttaaat caaattttaa tggagaaaat 3721 atttctggaa tatgttcaat aggtacttat cgttttagac ttggaggtga ttggtataga 3781 cacaattatt tggtgcctac tgtgaagcaa ggaaattatg cttcattatt agaatcaaca 3841 tcaactcatt ggggttttgt acctgtaagt gaataa c. LC / C: coordinates from crystal structure PDB:2QN0 • N-Term 1-17 • MLD-R1284-305 • MLD-R2306-342 • MLD-R1-R2283-343 • LHD-R1-R2-R3283-367 • LC / C11-437 • BoNT / C11291 . 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 NIFSKELSNE DINIVYEGQI QB\650053.01105\92299987.1 Page 71 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 1081 LRNVIKDYWG NPLKFDTEYY IINDNYIDRY IAPESNVLVL VQYPDRSKLY TGNPITIKSV 1141 SDKNPYSRIL NGDNIILHML YNSRKYMIIR DTDTIYATQG GECSQNCVYA LKLQSNLGNY 1201 GIGIFSIKNI VSKNKYCSQI FSSFRENTML LADIYKPWRF SFKNAYTPVA VTNYETKLLS 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 ctacttcaaa gtatcaaagt tattatgatc ctagttattt atctactgat 241 gaacaaaaag atacattttt aaaagggatt ataaaattat ttaaaagaat taatgaaaga 301 gatataggaa aaaaattaat aaattattta gtagttggtt caccttttat gggagattca 361 agtacgcctg aagatacatt tgattttaca cgtcatacta ctaatattgc agttgaaaag 421 tttgaaaatg gtagttggaa agtaacaaat attataacac caagtgtatt gatatttgga 481 ccacttccta atatattaga ctatacagca tcccttacat tgcaaggaca acaatcaaat 541 ccatcatttg aagggtttgg aacattatct atactaaaag tagcacctga atttttgtta 601 acatttagtg atgtaacatc taatcaaagt tcagctgtat taggcaaatc tatattttgt 661 atggatccag taatagcttt aatgcatgag ttaacacatt ctttgcatca attatatgga 721 ataaatatac catctgataa aaggattcgt ccacaagtta gcgagggatt tttctctcaa 781 gatggaccca acgtacaatt tgaggaatta tatacatttg gaggattaga tgttgaaata 841 atacctcaaa ttgaaagatc acaattaaga gaaaaagcat taggtcacta taaagatata 901 gcgaaaagac ttaataatat taataaaact attccttcta gttggattag taatatagat 961 aaatataaaa aaatattttc tgaaaagtat aattttgata aagataatac aggaaatttt 1021 gttgtaaata ttgataaatt caatagctta tattcagact tgactaatgt tatgtcagaa 1081 gttgtttatt cttcgcaata taatgttaaa aacaggactc attatttttc aaggcattat 1141 ctacctgtat ttgcaaatat attagatgat aatatttata ctataagaga tggttttaat 1201 ttaacaaata aaggttttaa tatagaaaat tcgggtcaga atatagaaag gaatcctgca 1261 ctacaaaagc ttagttcaga aagtgtagta gatttattta caaaagtatg tttaagatta 1321 acaaaaaata gtagagatga ttcaacatgt attaaagtta aaaataatag attaccttat 1381 gtagctgata aagatagcat ttcacaagaa atatttgaaa ataaaattat tacagatgag 1441 actaatgtac aaaattattc agataaattt tcattagatg aatctatttt agatgggcaa 1501 gttcctatta atcctgaaat agtagatcca ctattaccca atgttaatat ggaaccttta 1561 aatcttccag gtgaagaaat agtattttat gatgatatta ctaaatatgt tgattattta 1621 aattcttatt attatttgga atctcaaaaa ttaagtaata atgttgaaaa tattactctt 1681 acaacttcag ttgaagaagc attaggttat agcaataaga tatacacatt tttacctagc 1741 ttagctgaaa aagtgaataa aggtgttcaa gcaggtttat tcttaaattg ggcgaatgaa 1801 gtagttgagg attttactac aaatattatg aagaaagata cattggataa aatatcagat 1861 gtatcagtaa taattccata tataggacct gccttaaata taggaaattc agcattaagg 1921 ggaaatttta atcaagcatt tgcaacagct ggtgtagctt ttttattaga gggatttcca 1981 gagtttacta tacctgcact cggtgtattt accttttata gttctattca agaaagagag 2041 aaaattatta aaactataga aaattgtttg gaacaaagag ttaagagatg gaaagattca 2101 tatcaatgga tggtatcaaa ttggttgtca agaattacta ctcaatttaa tcatataaat 2161 tatcaaatgt atgattcttt aagttatcag gcagatgcaa tcaaagctaa aatagattta 2221 gaatataaaa aatactcagg aagtgataaa gaaaatataa aaagtcaagt tgaaaattta 2281 aaaaatagtt tagatgtaaa aatttcggaa gcaatgaata atataaataa atttatacga 2341 gaatgttctg taacatactt atttaaaaat atgctcccta aagtaattga cgaattaaat 2401 aagtttgatt taagaactaa aacagaatta attaatctta tagatagtca taatattatt 2461 ctagttggtg aagtagatag attaaaagca aaagtaaatg agagttttga aaatacaatg 2521 ccttttaata ttttttcata tactaataat tctttattaa aagatataat taatgaatat 2581 ttcaatagta ttaatgattc aaaaattttg agcttacaaa acaaaaaaaa tgctttagtg 2641 gatacatcag gatataatgc agaagtgagg gtaggagata atgttcaact taatacgata 2701 tatacaaatg actttaaatt aagtagttca ggagataaaa ttatagtaaa tttaaataat 2761 aatattttat atagcgctat ttatgagaac tctagtgtta gtttttggat taagatatct 2821 aaagatttaa ctaattctca taatgaatat acaataatta acagtataga acaaaattct 2881 gggtggaaat tatgtattag gaatggcaat atagaatgga ttttacaaga tgttaataga 2941 aagtataaaa gtttaatttt tgattatagt gaatcattaa gtcatacagg atatacaaat 3001 aaatggtttt ttgttactat aactaataat ataatggggt atatgaaact ttatataaat 3061 ggagaattaa agcagagtca aaaaattgaa gatttagatg aggttaagtt agataaaacc QB\650053.01105\92299987.1 Page 72 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 3121 atagtatttg gaatagatga gaatatagat gagaatcaga tgctttggat tagagatttt 3181 aatatttttt ctaaagaatt aagtaatgaa gatattaata ttgtatatga gggacaaata 3241 ttaagaaatg ttattaaaga ttattgggga aatcctttga agtttgatac agaatattat 3301 attattaatg ataattatat agataggtat atagcacctg aaagtaatgt acttgtactt 3361 gttcagtatc cagatagatc taaattatat actggaaatc ctattactat taaatcagta 3421 tctgataaga atccttatag tagaatttta aatggagata atataattct tcatatgtta 3481 tataatagta ggaaatatat gataataaga gatactgata caatatatgc aacacaagga 3541 ggagagtgtt cacaaaattg tgtatatgca ttaaaattac agagtaattt aggtaattat 3601 ggtataggta tatttagtat aaaaaatatt gtatctaaaa ataaatattg tagtcaaatt 3661 ttctctagtt ttagggaaaa tacaatgctt ctagcagata tatataaacc ttggagattt 3721 tcttttaaaa atgcatacac gccagttgca gtaactaatt atgaaacaaa actattatca 3781 acttcatctt tttggaaatt tatttctagg gatccaggat gggtagagta a   c. LC / D coordinates from crystal structure PDB:2FPQ • N-Term 1-18 • MLD-R1284-307 • MLD-R2308-342 • MLD-R1-R2284-342 • LHD-R1-R2-R3284-367 • 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 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 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 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 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 QB\650053.01105\92299987.1 Page 73 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 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 GIRYFKVFDT ELGKTEIETL 1081 YSDEPDPSIL KDFWGNYLLY NKRYYLLNLL RTDKSITQNS NFLNINQQRG VYQKPNIFSN 1141 TRLYTGVEVI IRKNGSTDIS NTDNFVRKND LAYINVVDRD VEYRLYADIS IAKPEKIIKL 1201 IRTSNSNNSL GQIIVMDSIG NNCTMNFQNN NGGNIGLLGF HSNNLVASSW YYNNIRKNTS 1261 SNGCFWSFIS KEHGWQEN b. DNA sequence BoNT / F (SEQ ID NO: 27) 1 atgccagttg taataaatag ttttaattat aatgaccctg ttaatgatga tacaatttta 61 tacatgcaga taccatatga agaaaaaagt aaaaaatatt ataaagcttt tgagattatg 121 cgtaatgttt ggataattcc tgagagaaat acaataggaa cggatcctag tgattttgat 181 ccaccggctt cattagagaa cggaagcagt gcttattatg atcctaatta tttaaccact 241 gatgctgaaa aagatagata tttaaaaaca acgataaaat tatttaagag aattaatagt 301 aatcctgcag gggaagtttt gttacaagaa atatcatatg ctaaaccata tttaggaaat 361 gaacacacgc caattaatga attccatcca gttactagaa ctacaagtgt taatataaaa 421 tcatcaacta atgttaaaag ttcaataata ttgaatcttc ttgtattggg agcaggacct 481 gatatatttg aaaattcttc ttaccccgtt agaaaactaa tggattcagg tggagtttat 541 gacccaagta atgatggttt tggatcaatt aatatcgtga cattttcacc tgaatatgaa 601 tatactttta atgatattag tggagggtat aacagtagta cagaatcatt tattgcagat 661 cctgcaattt cactagctca tgaattgata catgcactgc atggattata cggggctagg 721 ggagttactt ataaagagac tataaaagta aagcaagcac ctcttatgat agccgaaaaa 781 cccataaggc tagaagaatt tttaaccttt ggaggtcagg atttaaatat tattactagt 841 gctatgaagg aaaaaatata taacaatctt ttagctaact atgaaaaaat agctactaga 901 cttagtagag ttaatagtgc tcctcctgaa tatgatatta atgaatataa agattatttt 961 caatggaagt atgggctaga taaaaatgct gatggaagtt atactgtaaa tgaaaataaa 1021 tttaatgaaa tttataaaaa attatatagc tttacagaga ttgacttagc aaataaattt 1081 aaagtaaaat gtagaaatac ttattttatt aaatatggat ttttaaaagt tccaaatttg 1141 ttagatgatg atatttatac tgtatcagag gggtttaata taggtaattt agcagtaaac 1201 aatcgcggac aaaatataaa gttaaatcct aaaattattg attccattcc agataaaggt 1261 ctagtggaaa agatcgttaa attttgtaag agcgttattc ctagaaaagg tacaaaggcg 1321 ccaccgcgac tatgcattag agtaaataat agggagttat tttttgtagc ttcagaaagt 1381 agctataatg aaaatgatat taatacacct aaagaaattg acgatacaac aaatctaaat 1441 aataattata gaaataattt agatgaagtt attttagatt ataatagtga gacaatacct 1501 caaatatcaa atcaaacatt aaatacactt gtacaagacg atagttatgt gccaagatat 1561 gattctaatg gaacaagtga aatagaggaa cataatgttg ttgaccttaa tgtatttttc 1621 tatttacatg cacaaaaagt accagaaggt gaaactaata taagtttaac ttcttcaatt 1681 gatacggcat tatcagaaga atcgcaagta tatacattct tttcttcaga gtttattaat 1741 actatcaata aacctgtaca cgcagcacta tttataagtt ggataaatca agtaataaga 1801 gattttacta ctgaagctac acaaaaaagt acttttgata agattgcaga catatcttta 1861 gttgtaccat atgtaggtct tgctttaaat ataggtaatg aggtacaaaa agaaaatttt 1921 aaggaggcat ttgaattatt aggagcgggt attttattag aatttgtgcc agagctttta 1981 attcctacaa ttttagtgtt tacaataaaa tcctttatag gttcatctga gaataaaaat 2041 aaaatcatta aagcaataaa taattcatta atggaaagag aaacaaagtg gaaagaaata 2101 tatagttgga tagtatcaaa ttggcttact agaattaata cacaatttaa taaaagaaaa 2161 gaacaaatgt atcaagcttt gcaaaatcaa gtagatgcaa taaaaacagt aatagaatat 2221 aaatataata attatacttc agatgagaga aatagacttg aatctgaata taatatcaat 2281 aatataagag aagaattgaa caaaaaagtt tctttagcaa tggaaaatat agagagattt 2341 ataacagaga gttctatatt ttatttaatg aagttaataa atgaagccaa agttagtaaa 2401 ttaagagaat atgatgaagg cgttaaggaa tatttgctag actatatttc agaacataga 2461 tcaattttag gaaatagtgt acaagaatta aatgatttag tgactagtac tctgaataat 2521 agtattccat ttgaactttc ttcatatact aatgataaaa ttctaatttt atattttaat 2581 aaattatata aaaaaattaa agataactct attttagata tgcgatatga aaataataaa 2641 tttatagata tctctggata tggttcaaat ataagcatta atggagatgt atatatttat QB\650053.01105\92299987.1 Page 74 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 2701 tcaacaaata gaaatcaatt tggaatatat agtagtaagc ctagtgaagt taatatagct 2761 caaaataatg atattatata caatggtaga tatcaaaatt ttagtattag tttctgggta 2821 aggattccta aatacttcaa taaagtgaat cttaataatg aatatactat aatagattgt 2881 ataaggaata ataattcagg atggaaaata tcacttaatt ataataaaat aatttggact 2941 ttacaagata ctgctggaaa taatcaaaaa ctagttttta attatacaca aatgattagt 3001 atatctgatt atataaataa atggattttt gtaactatta ctaataatag attaggcaat 3061 tctagaattt acatcaatgg aaatttaata gatgaaaaat caatttcgaa tttaggtgat 3121 attcatgtta gtgataatat attatttaaa attgttggtt gtaatgatac aagatatgtt 3181 ggtataagat attttaaagt ttttgatacg gaattaggta aaacagaaat tgagacttta 3241 tatagtgatg agccagatcc aagtatctta aaagactttt ggggaaatta tttgttatat 3301 aataaaagat attatttatt gaatttacta agaacagata agtctattac tcagaattca 3361 aactttctaa atattaatca acaaagaggt gtttatcaga aaccaaatat tttttccaac 3421 actagattat atacaggagt agaagttatt ataagaaaaa atggatctac agatatatct 3481 aatacagata attttgttag aaaaaatgat ctggcatata ttaatgtagt agatcgtgat 3541 gtagaatatc ggctatatgc tgatatatca attgcaaaac cagagaaaat aataaaatta 3601 ataagaacat ctaattcaaa caatagctta ggtcaaatta tagttatgga ttcaatagga 3661 aataattgca caatgaattt tcaaaacaat aatgggggca atataggatt actaggtttt 3721 cattcaaata atttggttgc tagtagttgg tattataaca atatacgaaa aaatactagc 3781 agtaatggat gcttttggag ttttatttct aaagagcatg gatggcaaga aaactaa c. LC / F coordinate from crystal structure PDB:2A8A ^ N-Term 1-17 ^ MLD-R1281-302 ^ MLD-R2303-336 ^ MLD-R1-R2280-336 ^ LHD-R1-R2-R3283-360 ^ LC / F 1-429 ^ BoNT / F 1278 6. BoNT / G a. amino acid sequence LC / G 1-436 (SEQ ID NO: 28) 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 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 QB\650053.01105\92299987.1 Page 75 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 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 FVWIKDFNIF GRELNATEVS 1081 SLYWIQSSTN TLKDFWGNPL RYDTQYYLFN QGMQNIYIKY FSKASMGETA PRTNFNNAAI 1141 NYQNLYLGLR FIIKKASNSR NINNDNIVRE GDYIYLNIDN ISDESYRVYV LVNSKEIQTQ 1201 LFLAPINDDP TFYDVLQIKK YYEKTTYNCQ ILCEKDTKTF GLFGIGKFVK DYGYVWDTYD 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 aagaattttt 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 gctttactga aactaatcta 1081 gctggtgaat atggaataaa aactaggtat tcttatttta gtgaatattt gccaccgata 1141 aaaactgaaa aattgttaga caatacaatt tatactcaaa atgaaggctt taacatagct 1201 agtaaaaatc tcaaaacgga atttaatggt cagaataagg cggtaaataa agaggcttat 1261 gaagaaatca gcctagaaca tctcgttata tatagaatag caatgtgcaa gcctgtaatg 1321 tacaaaaata ccggtaaatc tgaacagtgt attattgtta ataatgagga tttatttttc 1381 atagctaata aagatagttt ttcaaaagat ttagctaaag cagaaactat agcatataat 1441 acacaaaata atactataga aaataatttt tctatagatc agttgatttt agataatgat 1501 ttaagcagtg gcatagactt accaaatgaa aacacagaac catttacaaa ttttgacgac 1561 atagatatcc ctgtgtatat taaacaatct gctttaaaaa aaatttttgt ggatggagat 1621 agcctttttg aatatttaca tgctcaaaca tttccttcta atatagaaaa tctacaacta 1681 acgaattcat taaatgatgc tttaagaaat aataataaag tctatacttt tttttctaca 1741 aaccttgttg aaaaagctaa tacagttgta ggtgcttcac tttttgtaaa ctgggtaaaa 1801 ggagtaatag atgattttac atctgaatcc acacaaaaaa gtactataga taaagtttca 1861 gatgtatcca taattattcc ctatatagga cctgctttga atgtaggaaa tgaaacagct 1921 aaagaaaatt ttaaaaatgc ttttgaaata ggtggagccg ctatcttaat ggagtttatt 1981 ccagaactta ttgtacctat agttggattt tttacattag aatcatatgt aggaaataaa 2041 gggcatatta ttatgacgat atccaatgct ttaaagaaaa gggatcaaaa atggacagat 2101 atgtatggtt tgatagtatc gcagtggctc tcaacggtta atactcaatt ttatacaata 2161 aaagaaagaa tgtacaatgc tttaaataat caatcacaag caatagaaaa aataatagaa 2221 gatcaatata atagatatag tgaagaagat aaaatgaata ttaacattga ttttaatgat QB\650053.01105\92299987.1 Page 76 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 2281 atagatttta aacttaatca aagtataaat ttagcaataa acaatataga tgattttata 2341 aaccaatgtt ctatatcata tctaatgaat agaatgattc cattagctgt aaaaaagtta 2401 aaagactttg atgataatct taagagagat ttattggagt atatagatac aaatgaacta 2461 tatttacttg atgaagtaaa tattctaaaa tcaaaagtaa atagacacct aaaagacagt 2521 ataccatttg atctttcact atataccaag gacacaattt taatacaagt ttttaataat 2581 tatattagta atattagtag taatgctatt ttaagtttaa gttatagagg tgggcgttta 2641 atagattcat ctggatatgg tgcaactatg aatgtaggtt cagatgttat ctttaatgat 2701 ataggaaatg gtcaatttaa attaaataat tctgaaaata gtaatattac ggcacatcaa 2761 agtaaattcg ttgtatatga tagtatgttt gataatttta gcattaactt ttgggtaagg 2821 actcctaaat ataataataa tgatatacaa acttatcttc aaaatgagta tacaataatt 2881 agttgtataa aaaatgactc aggatggaaa gtatctatta agggaaatag aataatatgg 2941 acattaatag atgttaatgc aaaatctaaa tcaatatttt tcgaatatag tataaaagat 3001 aatatatcag attatataaa taaatggttt tccataacta ttactaatga tagattaggt 3061 aacgcaaata tttatataaa tggaagtttg aaaaaaagtg aaaaaatttt aaacttagat 3121 agaattaatt ctagtaatga tatagacttc aaattaatta attgtacaga tactactaaa 3181 tttgtttgga ttaaggattt taatattttt ggtagagaat taaatgctac agaagtatct 3241 tcactatatt ggattcaatc atctacaaat actttaaaag atttttgggg gaatccttta 3301 agatacgata cacaatacta tctgtttaat caaggtatgc aaaatatcta tataaagtat 3361 tttagtaaag cttctatggg ggaaactgca ccacgtacaa actttaataa tgcagcaata 3421 aattatcaaa atttatatct tggtttacga tttattataa aaaaagcatc aaattctcgg 3481 aatataaata atgataatat agtcagagaa ggagattata tatatcttaa tattgataat 3541 atttctgatg aatcttacag agtatatgtt ttggtgaatt ctaaagaaat tcaaactcaa 3601 ttatttttag cacccataaa tgatgatcct acgttctatg atgtactaca aataaaaaaa 3661 tattatgaaa aaacaacata taattgtcag atactttgcg aaaaagatac taaaacattt 3721 gggctgtttg gaattggtaa atttgttaaa gattatggat atgtttggga tacctatgat 3781 aattattttt gcataagtca gtggtatctc agaagaatat ctgaaaatat aaataaatta 3841 aggttgggat gtaattggca attcattccc gtggatgaag gatggacaga ataa c. LC / G coordinates from crystal structure PDB:1ZB7 ^ N-Term 1-18 ^ MLD-R1283-304 ^ MLD-R2305-339 ^ MLD-R1-R2283-339 ^ LHD-R1-R2-R3283-365 ^ LC / G 1-436 ^ BoNT / G 1297 ^ 7. BoNT / A3 a. amino acid sequence LC / A31-426 (SEQ ID NO: 31) 1 MPFVNKQFNY RDPVNGVDIA 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 ISVNKPAFKE FYRVLTRGFT ELEFVNPFKV 361 INRKTYLNFD KAVFRINIVP DENYTINEGF NLEGANSNGQ NTEINSRNFT RLKNFTGLF 421 FYKLLC QB\650053.01105\92299987.1 Page 77 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION b. amino acid sequence BoNT / A31-1292 (SEQ ID NO: 32) 1 MPFVNKQFNY RDPVNGVDIA 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 ISVNKPAFKE FYRVLTRGFT ELEFVNPFKV 361 INRKTYLNFD KAVFRINIVP DENYTINEGF NLEGANSNGQ NTEINSRNFT RLKNFTGLFE 421 FYKLLCVRGI IPFKTKSLDE GYNKALNDLC 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 RYIMIKYFNL FDKELNEKEI 1081 KDLYDSQSNP GILKDFWGNY LQYDKPYYML NLFDPNKYVD VNNIGIRGYM YLKGPRGSVM 1141 TTNIYLNSTL YMGTKFIIKK YASGNEDNIV RNNDRVYINV VVKNKEYRLA TNASQAGVEK 1201 ILSALEIPDV GNLSQVVVMK SKDDQGIRNK CKMNLQDNNG NDIGFIGFHL YDNIAKLVAS 1261 NWYNRQVGKA SRTFGCSWEF IPVDDGWGES SL b. DNA sequence BoNT / A3 (SEQ ID NO: 33) 1 atgccatttg ttaataaaca atttaattat agagatcctg taaatggtgt 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 ggttctactc aatacattag atttagccca gattttacat ttggttttga ggagtcactt 601 gaagttgata caaatcctct tttaggtgca ggcacatttg ctacagatcc agcggtaaca 661 ttagcacatg aacttataca tgctgcacat agattatatg gaatagcaat taatccaaat 721 agggttttaa aagtaaagac taatgcctat tatgaaatga gtgggttaga agtaagcttt 781 gaggaactta gaacatttgg gggaaatgat acaaacttta tagatagttt atggcaaaaa 841 aaatttagta gagacgctta tgataatctt caaaatatag cacgtatact taatgaagct 901 aaaacaatag taggtactac tactccatta cagtatatga aaaatatttt tatacggaaa QB\650053.01105\92299987.1 Page 78 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 961 tatttcttat ctgaagatgc atctggaaag atttcggtaa ataaaccagc atttaaggaa 1021 ttttacaggg tgttaacaag gggtttcaca gagttagaat ttgttaatcc ttttaaagta 1081 attaacagga aaacatattt gaattttgat aaagccgtat ttaggataaa tatagtacct 1141 gatgaaaatt acacaataaa tgagggattt aatttagaag gtgcaaattc taatggtcag 1201 aatacagaaa ttaatagtag gaattttact agattaaaaa attttacagg attatttgaa 1261 ttttataagc tgctatgtgt aagagggata atacctttta aaactaaatc attagatgaa 1321 ggatacaata aggcattaaa tgatttatgt atcaaagtta ataattggga cttgtttttt 1381 agtccttcag aagataattt tactaatgat ttagataaag tagaagaaat tacagctgat 1441 accaatatag aagcagcaga agaaaatatt agttcagatt taatacaaca atattattta 1501 acttttgatt ttgataatga acctgaaaat atttcaatag aaaatctttc aagtgatatt 1561 ataggccaat tagagcctat gcctaatata gaaagatttc ccaatggaaa aaagtatgag 1621 ttagataaat atactatgtt ccattatctt cgtgctcaag aatttgaaca tggtgattct 1681 agaattatct taacaaattc tgctgaagaa gcattattaa agcctaatgt tgcttacaca 1741 tttttttctt caaaatatgt aaagaagatt aataaagccg tagaggcagt tattttttta 1801 agttgggcag aagagttagt atatgacttt accgatgaaa ctaatgaagt aactactatg 1861 gataaaattg ctgatataac tataattgtt ccatatatag ggcctgcttt aaatataggt 1921 aatatggtat ctaaaggaga gttcgtagaa gctatactat ttacgggagt tgttgctctg 1981 ttagaattta taccagagta ttccctccct gtatttggta cttttgcaat tgtatcatat 2041 attgccaata aggttctaac tgttcaaaca ataaataatg ctttaagtaa aagaaatgaa 2101 aaatgggatg aagtctataa atatacagta acaaattggc tagcaaaggt taatacacag 2161 attgatctaa taagggaaaa aatgaaaaaa gctttagaaa atcaggcaga agcaacaagg 2221 gctataataa actatcagta taatcaatat actgaggaag agaaaaataa tattaatttt 2281 aatattgatg atttaagttc gaaacttaat aggtctataa atagggctat gattaacata 2341 aataaatttt tggatcaatg ctctgtttca tatttaatga attctatgat accttatgct 2401 gttaaacggt taaaagattt tgatgctagt gttagagatg tattattaaa gtatatatat 2461 gataatagag gaactttaat tcttcaagta gatagattaa aagatgaagt taataataca 2521 cttagtgcag atataccttt tcagctttct aaatacgtaa atgataaaaa attattatct 2581 acatttactg aatatattaa gaatattgtt aatacctcta tattgagtat agtatataaa 2641 aaagatgatt taatagattt atctaggtat ggagcaaaaa taaatattgg cgatagagta 2701 tattatgatt caatagataa aaatcaaatt aaattaatta atttagaaag tagtacaatt 2761 gaggtaattt taaaaaatgc tattgtatat aatagtatgt atgaaaattt tagtactagc 2821 ttttggataa aaattcctaa gtattttagc aagataaatc taaataatga atatacaata 2881 ataaattgta tagaaaataa ttcaggatgg aaagtatcac ttaattatgg tgaaataatc 2941 tggactttgc aggataataa gcaaaacata caaagagtag tttttaaata cagtcaaatg 3001 gttaatatat cagattatat aaacagatgg atgtttgtaa ctatcactaa taatagacta 3061 actaaatcta aaatttatat aaatggaaga ttaatagatc aaaaaccaat ttcaaatttg 3121 ggtaatattc atgctagtaa taagataatg tttaaattag atggctgtag agatccacgt 3181 agatacatca tgataaaata tttcaatctt ttcgataaag aattaaatga aaaagaaatc 3241 aaagatttat atgatagtca atcaaatcca ggtattttaa aagacttttg gggtaattat 3301 ttacaatatg ataaaccata ctatatgtta aatttatttg atccaaataa atatgtcgat 3361 gtaaataata taggtattag aggttatatg tatcttaaag ggcctagagg tagcgtaatg 3421 actacaaaca tttatttaaa ttcaactttg tatatgggga caaaatttat tataaaaaaa 3481 tatgcttctg gaaatgaaga taatattgtt agaaataatg atcgtgtata tattaatgta 3541 gtagttaaaa ataaagaata taggttagct actaatgcat cacaggcagg cgtagaaaaa QB\650053.01105\92299987.1 Page 79 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 3601 atactaagtg cattagaaat acctgatgta ggaaatctaa gtcaagtagt agtaatgaag 3661 tcaaaagatg atcaaggaat aagaaataaa tgcaaaatga atttacaaga taataatggg 3721 aatgatatag gctttatagg atttcatttg tatgataata tagctaaact agtagcaagt 3781 aattggtata atagacaagt gggaaaagct agtaggactt tcggttgttc atgggagttt 3841 attcctgtag atgatggatg gggagaaagt tcactgtaa c. LC / A3 coordinates from crystal structure PDB:7DVL • N-Term 1-18 • MLD-R1277-300 • MLD-R2300-334 • MLD-R1-R2277-334 • LHD-R1-R2-R3277-335-359 • LC / A31-426 • BoNT / A31292 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 RLKNFTGLFE 421 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 FYKLLCVRGI 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 QB\650053.01105\92299987.1 Page 80 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 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 RYIMIKYFNL FDKELNEKEI 1081 KDLYDSQSNP GILKDFWGNY LQYDKPYYML NLFDPNKYVD VNNIGIRGYM YLKGPRGSVM 1141 TTNIYLNSTL YMGTKFIIKK YASGNEDNIV RNNDRVYINV VVKNKEYRLA TNASQAGVEK 1201 ILSALEIPDV GNLSQVVVMK SKDDQGIRNK CKMNLQDNNG NDIGFVGFHL YDNIAKLVAS 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 ggttctactc aatacattag atttagccca gattttacat ttggttttga ggagtcactt 601 gaagttgata caaatcctct tttaggtgca ggcacatttg ctacagatcc agcggtaaca 661 ttagcacatg aacttataca tgctgcacat agattatatg gaatagcaat taatccaaat 721 agggttttaa aagtaaagac taatgcctat tatgaaatga gtgggttaga agtaagcttt 781 gaggaactta gaacatttgg gggaaatgat acaaacttta tagatagttt atggcaaaaa 841 aaatttagta gagacgctta tgataatctt caaaatatag cacgtatact taatgaagct 901 aaaacaatag taggtactac tactccatta cagtatatga aaaatatttt tatacggaaa 961 tatttcttat ctgaagatgc atctggaaag atttcggtaa ataaagcagc atttaaggaa 1021 ttttacaggg tgttaacaag gggtttcaca gagttagaat ttgttaatcc ttttaaagta 1081 attaacagaa aaacatattt gaattttgat aaagccgtat ttaggataaa tatagtacct 1141 gatgaaaatt acacaataaa tgagggattt aatttagaag gtgcaaattc taatggtcag 1201 aatacagaaa ttaatagtag gaattttact agattaaaaa attttacagg attatttgaa 1261 ttttataagc tgctatgtgt aagagggata atacctttta aaactaaatc attagatgaa 1321 ggatacaata aggcattaaa ttatttatgt atcaaagtta ataattggga cttgtttttt 1381 agtccttcag aagataattt tactaatgat ttagataaag tagaagaaat tacagctgat 1441 accaatatag aagcagcaga agaaaatatt agttcagatt taatacaaca atattattta 1501 acttttgatt ttgataatga acctgaaaat atttcaatag aaaatctttc aagtgatatt 1561 ataggccaat tagagcctat gcctaatata gaaagatttc ccaatggaaa aaagtatgag 1621 ttagataaat atactatgtt ccattatctt cgtgctcaag aatttgaaca tggtgattct 1681 agaattatct taacaaattc tgctgaagaa gcattattaa agcctaatgt tgcttacaca 1741 tttttttctt caaaatatgt aaagaagatt aataaagccg tagaggcagt tattttttta 1801 agttgggcag aagagttagt atatgacttt accgatgaaa ctaatgaagt aactactatg 1861 gataaaattg ctgatataac tataattgtt ccatatatag ggcctgcttt aaatataggt 1921 aatatggtat ctaaaggaga gttcgtagaa gctatactat ttacgggagt tgttgctctg 1981 ttagaattta taccagagta ttccctccct gtatttggta cttttgcaat tgtatcatat 2041 attgccaata aggttctaac tgttcaaaca ataaataatg ctttaagtaa aagaaatgaa 2101 aaatgggatg aagtctataa atatacagta acaaattggc tagcaaaggt taatacacag 2161 attgatctaa taagggaaaa aatgaaaaaa gctttagaaa atcaggcaga agcaacaagg 2221 gctataataa actatcagta taatcaatat actgaggaag agaaaaataa tattaatttt 2281 aatattgatg atttaagttc gaaacttaat aggtctataa atagggctat gattaacata 2341 aataaatttt tggatcaatg ctctgtttca tatttaatga attctatgat accttatgct 2401 gttaaacggt taaaagattt tgatgctagt gttagagatg tattattaaa gtatatatat 2461 gataatagag gaactttaat tcttcaagta gatagattaa aagatgaagt taataataca 2521 cttagtgcag atataccttt tcagctttct aaatacgtaa atgataaaaa attattatct 2581 acatttactg aatatattaa gaatattgtt aatacctcta tattgagtat agtatataaa 2641 aaagatgatt taatagattt atctaggtat ggagcaaaaa taaatattgg cgatagagta 2701 tattatgatt caatagataa aaatcaaatt aaattaatta atttagaaag tagtacaatt QB\650053.01105\92299987.1 Page 81 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 2761 gaggtaattt taaaaaatgc tattgtatat aatagtatgt atgaaaattt tagtactagc 2821 ttttggataa aaattcctaa gtattttagc aagataaatc taaataatga atatacaata 2881 ataaattgta tagaaaataa ttcaggatgg aaagtatcac ttaattatgg tgaaataatc 2941 tggactttgc aggataataa gcaaaacata caaagagtag tttttaaata cagtcaaatg 3001 gttaatatat cagattatat aaacagatgg atgtttgtaa ctatcactaa taatagacta 3061 actaaatcta aaatttatat aaatggaaga ttaatagatc aaaaaccaat ttcaaatttg 3121 ggtaatattc atgctagtaa taagataatg tttaaattag atggctgtag agatccacgt 3181 agatacatca tgataaaata tttcaatctt ttcgataaag aattaaatga aaaagaaatc 3241 aaagatttat atgatagtca atcaaatcca ggtattttaa aagacttttg gggtaattat 3301 ttacaatatg ataaaccata ctatatgtta aatttatttg atccaaataa atatgtcgat 3361 gtaaataata taggtattag aggttatatg tatcttaaag ggcctagagg tagcgtaatg 3421 actacaaaca tttatttaaa ttcaactttg tatatgggga caaaatttat tataaaaaaa 3481 tatgcttctg gaaatgaaga taatattgtt agaaataatg atcgtgtata tattaatgta 3541 gtagttaaaa ataaagaata taggttagct actaatgcat cacaggcagg cgtagaaaaa 3601 atactaagtg cattagaaat acctgatgta ggaaatctaa gtcaagtagt agtaatgaag 3661 tcaaaagatg atcaaggaat aagaaataaa tgcaaaatga atttacaaga taataatggg 3721 aatgatatag gctttgtagg atttcatttg tatgataata tagctaaact agtagcaagt 3781 aattggtata atagacaagt gggaaaagct agtaggactt tcggttgttc atgggagttt 3841 attcctgtag atgatggatg gggagaaagt tcactgtaa c. LC / A3(V) coordinates from crystal structure PBD:7DVL (LC / A3) • N-Term 1-18 • MLD-R1277-299 • MLD-R2299-334 • MLD-R1-R2277-334 • LHD-R1-R2-R3277-335-359 • LC / A31-426 • BoNT / A31292 9. BoNT / A1 a. amino acid sequence BoNT / A11-1296 (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 QB\650053.01105\92299987.1 Page 82 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 1021 NNRLNNSKIY INGRLIDQKP ISNLGNIHAS NNIMFKLDGC RDTHRYIWIK YFNLFDKELN 1081 EKEIKDLYDN QSNSGILKDF WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR 1141 GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA 1201 GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI GFHQFNNIAK 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 AGGACAAATG CAACCAGTAA AAGCTTTTAA AATTCATAAT 121 AAAATATGGG TTATTCCAGA AAGAGATACA TTTACAAATC CTGAAGAAGG AGATTTAAAT 181 CCACCACCAG AAGCAAAACA AGTTCCAGTT TCATATTATG ATTCAACATA TTTAAGTACA 241 GATAATGAAA AAGATAATTA TTTAAAGGGA GTTACAAAAT TATTTGAGAG AATTTATTCA 301 ACTGATCTTG GAAGAATGTT GTTAACATCA ATAGTAAGGG GAATACCATT TTGGGGTGGA 361 AGTACAATAG ATACAGAATT AAAAGTTATT GATACTAATT GTATTAATGT GATACAACCA 421 GATGGTAGTT ATAGATCAGA AGAACTTAAT CTAGTAATAA TAGGACCCTC AGCTGATATT 481 ATACAGTTTG AATGTAAAAG CTTTGGACAT GAAGTTTTGA ATCTTACGCG AAATGGTTAT 541 GGCTCTACTC AATACATTAG ATTTAGCCCA GATTTTACAT TTGGTTTTGA GGAGTCACTT 601 GAAGTTGATA CAAATCCTCT TTTAGGTGCA GGCAAATTTG CTACAGATCC AGCAGTAACA 661 TTAGCACATG AACTTATACA TGCTGGACAT AGATTATATG GAATAGCAAT TAATCCAAAT 721 AGGGTTTTTA AAGTAAATAC TAATGCCTAT TATGAAATGA GTGGGTTAGA AGTAAGCTTT 781 GAGGAACTTA GAACATTTGG GGGACATGAT GCAAAGTTTA TAGATAGTTT ACAGGAAAAC 841 GAATTTCGTC TATATTATTA TAATAAGTTT AAAGATATAG CAAGTACACT TAATAAAGCT 901 AAATCAATAG TAGGTACTAC TGCTTCATTA CAGTATATGA AAAATGTTTT TAAAGAGAAA 961 TATCTCCTAT CTGAAGATAC ATCTGGAAAA TTTTCGGTAG ATAAATTAAA ATTTGATAAG 1021 TTATACAAAA TGTTAACAGA GATTTACACA GAGGATAATT TTGTTAAGTT TTTTAAAGTA 1081 CTTAACAGAA AAACATATTT GAATTTTGAT AAAGCCGTAT TTAAGATAAA TATAGTACCT 1141 AAGGTAAATT ACACAATATA TGATGGATTT AATTTAAGAA ATACAAATTT AGCAGCAAAC 1201 TTTAATGGTC AAAATACAGA AATTAATAAT ATGAATTTTA CTAAACTAAA AAATTTTACT 1261 GGATTGTTTG AATTTTATAA GTTGCTATGT GTAAGAGGGA TAATAACTTC TAAAACTAAA 1321 TCATTAGATA AAGGATACAA TAAGGCATTA AATGATTTAT GTATCAAAGT TAATAATTGG 1381 GACTTGTTTT TTAGTCCTTC AGAAGATAAT TTTACTAATG ATCTAAATAA AGGAGAAGAA 1441 ATTACATCTG ATACTAATAT AGAAGCAGCA GAAGAAAATA TTAGTTTAGA TTTAATACAA 1501 CAATATTATT TAACCTTTAA TTTTGATAAT GAACCTGAAA ATATTTCAAT AGAAAATCTT 1561 TCAAGTGACA TTATAGGCCA ATTAGAACTT ATGCCTAATA TAGAAAGATT TCCTAATGGA 1621 AAAAAGTATG AGTTAGATAA ATATACTATG TTCCATTATC TTCGTGCTCA AGAATTTGAA 1681 CATGGTAAAT CTAGGATTGC TTTAACAAAT TCTGTTAACG AAGCATTATT AAATCCTAGT 1741 CGTGTTTATA CATTTTTTTC TTCAGACTAT GTAAAGAAAG TTAATAAAGC TACGGAGGCA 1801 GCTATGTTTT TAGGCTGGGT AGAACAATTA GTATATGATT TTACCGATGA AACTAGCGAA 1861 GTAAGTACTA CGGATAAAAT TGCGGATATA ACTATAATTA TTCCATATAT AGGACCTGCT 1921 TTAAATATAG GTAATATGTT ATATAAAGAT GATTTTGTAG GTGCTTTAAT ATTTTCAGGA 1981 GCTGTTATTC TGTTAGAATT TATACCAGAG ATTGCAATAC CTGTATTAGG TACTTTTGCA 2041 CTTGTATCAT ATATTGCGAA TAAGGTTCTA ACCGTTCAAA CAATAGATAA TGCTTTAAGT 2101 AAAAGAAATG AAAAATGGGA TGAGGTCTAT AAATATATAG TAACAAATTG GTTAGCAAAG 2161 GTTAATACAC AGATTGATCT AATAAGAAAA AAAATGAAAG AAGCTTTAGA AAATCAAGCA 2221 GAAGCAACAA AGGCTATAAT AAACTATCAG TATAATCAAT ATACTGAGGA AGAGAAAAAT 2281 AATATTAATT TTAATATTGA TGATTTAAGT TCGAAACTTA ATGAGTCTAT AAATAAAGCT 2341 ATGATTAATA TAAATAAATT TTTGAATCAA TGCTCTGTTT CATATTTAAT GAATTCTATG 2401 ATCCCTTATG GTGTTAAACG GTTAGAAGAT TTTGATGCTA GTCTTAAAGA TGCATTATTA 2461 AAGTATATAT ATGATAATAG AGGAACTTTA ATTGGTCAAG TAGATAGATT AAAAGATAAA 2521 GTTAATAATA CACTTAGTAC AGATATACCT TTTCAGCTTT CCAAATACGT AGATAATCAA 2581 AGATTATTAT CTACATTTAC TGAATATATT AAGAATATTA TTAATACTTC TATATTGAAT 2641 TTAAGATATG AAAGTAATCA TTTAATAGAC TTATCTAGGT ATGCATCAAA AATAAATATT 2701 GGTAGTAAAG TAAATTTTGA TCCAATAGAT AAAAATCAAA TTCAATTATT TAATTTAGAA 2761 AGTAGTAAAA TTGAGGTAAT TTTAAAAAAT GCTATTGTAT ATAATAGTAT GTATGAAAAT 2821 TTTAGTACTA GCTTTTGGAT AAGAATTCCT AAGTATTTTA ACAGTATAAG TCTAAATAAT 2881 GAATATACAA TAATAAATTG TATGGAAAAT AATTCAGGAT GGAAAGTATC ACTTAATTAT 2941 GGTGAAATAA TCTGGACTTT ACAGGATACT CAGGAAATAA AACAAAGAGT AGTTTTTAAA QB\650053.01105\92299987.1 Page 83 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 3001 TACAGTCAAA TGATTAATAT ATCAGATTAT ATAAACAGAT GGATTTTTGT AACTATCACT 3061 AATAATAGAT TAAATAACTC TAAAATTTAT ATAAATGGAA GATTAATAGA TCAAAAACCA 3121 ATTTCAAATT TAGGTAATAT TCATGCTAGT AATAATATAA TGTTTAAATT AGATGGTTGT 3181 AGAGATACAC ATAGATATAT TTGGATAAAA TATTTTAATC TTTTTGATAA GGAATTAAAT 3241 GAAAAAGAAA TCAAAGATTT ATATGATAAT CAATCAAATT CAGGTATTTT AAAAGACTTT 3301 TGGGGTGATT ATTTACAATA TGATAAACCA TACTATATGT TAAATTTATA TGATCCAAAT 3361 AAATATGTCG ATGTAAATAA TGTAGGTATT AGAGGTTATA TGTATCTTAA AGGGCCTAGA 3421 GGTAGCGTAA TGACTACAAA CATTTATTTA AATTCAAGTT TGTATAGGGG GACAAAATTT 3481 ATTATAAAAA AATATGCTTC TGGAAATAAA GATAATATTG TTAGAAATAA TGATCGTGTA 3541 TATATTAATG TAGTAGTTAA AAATAAAGAA TATAGGTTAG CTACTAATGC ATCACAGGCA 3601 GGCGTAGAAA AAATACTAAG TGCATTAGAA ATACCTGATG TAGGAAATCT AAGTCAAGTA 3661 GTAGTAATGA AGTCAAAAAA TGATCAAGGA ATAACAAATA AATGCAAAAT GAATTTACAA 3721 GATAATAATG GGAATGATAT AGGCTTTATA GGATTTCATC AGTTTAATAA TATAGCTAAA 3781 CTAGTAGCAA GTAATTGGTA TAATAGACAA ATAGAAAGAT CTAGTAGGAC TTTGGGTTGC 3841 TCATGGGAAT TTATTCCTGT AGATGATGGA TGGGGAGAAA GGCCACTGTA A

[0307] Example 5

[0308] Botulinum neurotoxin serotype A1 (BoNT / A1) is among the most potent human protein toxins, but also an important human therapy. BoNT / A1 comprises a catalytic Light Chain (LC / A1) linked by a disulfide bond to a Heavy Chain. How intracellular LC / A1 localizes to the plasma membrane to cleave Synaptosomal Associated Protein 25 (SNAP-25) is unknown. Imaging analysis showed LC / A1 migrated on microtubules and colocalized with several Rab GTPases. Using functionally diverse DN Rab GTPases, for the first time LC / A1 is shown to use a fast, synaptic vesicle recycling pathway to traffic and cleave plasma membrane bound SNAP-25. Mechanistic studies showed Light Chain stable intracellular localization defined LC / A potency and duration of action. These outcomes allow custom engineering of novel BoNT therapies and identification of new groups of host protein target to limit BoNT intoxication.

[0309] Botulinum neurotoxins (BoNT) are Category A Tier 1 Select Agents and potential bioweapons due to their high potency and the severity and long duration of botulism in humans and vertebrate animals (1). There are no licensed human vaccines against botulism and a limited therapy window with heptavalent equine antiserum (BAT), which is effective only when administered early in the disease before BoNT enters motor neurons. Once inside neurons, BoNT persists and cleaves membrane bound v- or t- SNARE proteins, resulting in long-lasting (weeks to several months) flaccid paralysis characteristic of botulism (1). Each botulism outbreak is a reportable public health emergency (2).

[0310] BoNT are a structurally conserved family of proteins that comprise a catalytic Light Chain (LC), a zinc metalloprotease, linked by a disulfide bond to a Heavy Chain (HC). HC mediates binding to host cell receptors and LC translocation into the cytosol of a motor neuron. BoNT comprise seven immunologically distinct serotypes (A-G) and over 40 subtypes, where QB\650053.01105\92299987.1 Page 84 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION BoNT / A comprises 8 subtypes (3). One subtype, BoNT / A1 is an effective and widely used human therapy to treat numerous neuromuscular diseases (4). While BoNT / A1 entry and LC / A1 translocation into the neuronal cytosol have been addressed, the molecular mechanisms for LC / A1 intracellular trafficking to cleave the plasma membrane bound t-SNARE synaptosome associated protein of 25-kDa (SNAP-25) remains unsolved. In general, how catalytic components of protein toxins locate their intracellular targets is poorly understood.

[0311] Characterization of BoNT / A subtypes identified BoNT / A3 as having a short duration of action that was a property of the LC (1). While LC / A3 localized on intracellular vesicles, LC / A1 used two steps to stably localize to the plasma membrane of Neuro-2A (N2A) cells, a mouse neuroblastoma cell line (5). LC / A1 N terminus (termed A1 N, residues 1-17) mediated LC binding to intercellular vesicles, and an internal encoded membrane localization domain (termed MLD, residues 275-334) mediated LC / A1 stable localization to the plasma membrane (5–8). In a mouse model of botulism, LC mediated vesicle association and stable plasma membrane association contributed to BoNT / A1 high potency (9).

[0312] Targeted intracellular vesicle trafficking is aided by vesicle-bound Rab GTPases, members of the Ras superfamily of small G proteins (10). Thus, Rab GTPases can be used as molecular markers for vesicle trafficking pathways (11). However, within the neuronal bouton intracellular functions and localization of Rab GTPases often overlap, requiring the characterization of multiple Rab GTPases to resolve intracellular trafficking pathways (12). Using a subset of dominant negative (DN) Rab GTPases and LC / A1 and LC / A3 subtypes, LC / A1 was found traffic to the plasma membrane and cleave SNAP-25 by a fast branch of the synaptic vesicle recycling pathway. The stable membrane association of LC / A1 correlated with high potency and long duration of action.

[0313] Microtubules mediate LC / A1 intracellular trafficking in N2A cells

[0314] Microtubules and actin are critical components of intracellular movements, including cytokinesis, transport, and motility, and some microbial pathogens use microtubules and actin for intracellular trafficking (13, 14). Two cytoskeleton inhibitors tested a role for actin and / or microtubules in LC / A1 intracellular localization in N2A cells: cytochalasin D, which binds to polymerizing ends of actin filaments to disrupt actin extension (15, 16), and nocodazole, which binds ^-tubulin leading to microtubule depolymerization (17). N2A cells were transfected overnight with plasmids encoding EGFP-LC / A1, a membrane localized protein, or EGFP LC / A3, a vesicle localized protein. Cells were then treated with cytochalasin D or nocodazole and stained for actin (phalloidin) and for microtubules (β -tubulin) (15, 16). Cytochalasin D treatment of N2A cells caused actin disruption and neurite sprouts containing QB\650053.01105\92299987.1 Page 85 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION microtubules (β-tubulin positive fibers). In these cells, LC / A1 was retained on the plasma membrane, while LC / A3 was retained on intracellular vesicles Fig.15A. Thus, actin disruption did not alter ectopically expressed LC / A1 localization on the plasma membrane or LC / A3 vesicle association. Time-lapse immunofluorescence imaging of CD-treated N2A cells showed membrane localized LC / A1 and bi-directional trafficking of LC / A1 in neurite sprouts Data not shown. In contrast, N2A cells treated with nocodazole ectopically expressed LC / A1 on the plasma membrane and shifted LC / A3 from intracellular vesicles to the cytosol Fig. 15A. Together, these data showed that microtubules contribute to LC / A association with intracellular vesicles and intracellular trafficking to the plasma membrane.

[0315] LC / A1 colocalizes with Rab GTPases in N2A cells

[0316] The above results indicated LC / A uses microtubules for intracellular trafficking. Due to the complexity of neuronal vesicle pathways (11), a suite of Rab GTPases, which aid in vesicle compartmentalization and function in synaptic vesicle migration and / or cycling (18), were used to study LC / A1 intracellular trafficking Fig. 15B. At steady state, LC / A1 had the highest average Pearson’s Colocalization Coefficient (PCC) with Rab4, a marker of fast- recycling endosomes and Rab27a, a marker of late synaptic vesicles (SV) (19). LC / A1 showed an intermediate PCC with Rab5, a clathrin-dependent endocytosis marker that is also necessary for SV formation (11, 20), Rab11, a slow recycling and early sorting endosome marker (19), and Rab7, a late endosome marker (21). LC / A1 showed low colocalization with Rab3a, a late SV marker (19) Fig. 15C. Analyses of the intracellular compartments in individual cells showed co-localization of LC / A1 with Rab3a, Rab27a, and Rab4 along the plasma membrane, with Rab5, and Rab11 in the cell interior, and with Rab7 in the perinuclear region of N2A cells Fig.15B. Together, at steady state, LC / A1 colocalized with Rab GTPases involved in several intracellular functions, including the recycling endosomes, synaptic vesicle cycling, and retrograde trafficking endosomes (22).

[0317] Dominant negative (DN) Rab GTPases inhibit N-terminal mediated LC / A1 stable plasma membrane localization

[0318] DN Rab GTPases sequester Rho guanine nucleotide exchange factor (GEF), interfering with Rab GTPase-nucleotide exchange that halts Rab GTPase-directed vesicle movement (12, 23). Vesicle interactions that mediate LC / A1 intracellular trafficking to the plasma membrane were identified by co-transfection of 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 Fig. 16A. However, co-expression with neither DN Rab7 nor Rab11 altered LC / A1 plasma QB\650053.01105\92299987.1 Page 86 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION membrane localization indicating that neither Rab7 nor Rab11 alone are required for LC / A1 membrane localization. The observed inhibition of LC / A1 trafficking by DN Rab3a, DN Rab4, DN Rab5, and DN Rab27b implicated the fast SV recycling pathway for LC / A1 trafficking to the plasma membrane. LC / A3V (A1-MLD), a cytosolic LC / A3 chimera that shows a low efficiency localization to the plasma membrane independent of intracellular vesicles, (9), was tested to establish if LC / A-vesicle association was required to observe inhibition of LC / A trafficking to the plasma membrane by DN Rab GTPases Fig.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 membrane localization of LC / A3V (A1-MLD) independent of intracellular vesicles (9). These data also supported a role for vesicle mediated the fast SV recycling pathway in LC / A1 trafficking to the plasma membrane. Specificity of DN Rab GTPase function was validated assessing cholera toxin B subunit (CT / B) and transferrin intracellular trafficking in N2A cells by co-expression with individual DN Rab GTPases (25, 26) Fig.18. Co-expression of DN Rab4 or DN Rab11 had limited effects on the intracellular uptake and recycling of transferrin, and CT / B endocytosis was not inhibited by DN Rab3a, DN Rab4, DN Rab7, DN Rab11, or DN Rab27b, which function downstream of endocytosis (11). Overall, these experiments implicated a role for the N-terminus of LC / A1 in the fast SV recycling pathway for LC / A1 to traffic to the plasma membrane.

[0319] DN Rab GTPases inhibit N-terminal mediated LC / A1 cleavage of plasma membrane bound SNAP-25

[0320] While inhibition of LC / A1 plasma membrane localization by DN Rab GTPases defined bulk LC / A1 intracellular trafficking, experiments next tested the ability of the DN Rab GTPases to inhibit LC / A1 cleavage of SNAP-25. N2A transfection conditions with pEGFP- LC / A1were established to achieve an intracellular dose of LC / A1 that cleaved ~30% of total intracellular SNAP-25 as detected by Western blot Fig. 19A-B. Imaging individual cells transfected with pEGFP-LC / A1 alone had a range of LC / A1 expressions between 1 x10- 6 x10 sum intensity fluorescence that was proportional to the amount of cleaved SNAP-25 Fig 19E. Co-transfection of pEGFP-LC / A1 with individual plasmids encoding the DN Rab GTPases, where expression was confirmed by Western blot Fig.19C, showed LC / A1 cleavage of SNAP-25 was statistically inhibited by co-expression of DN Rab27b, DN Rab5, DN Rab3a, or DN Rab4, while coexpression of neither DN Rab7 nor DN Rab11 statistically inhibited LC / A1 cleavage of SNAP-25 Fig.19E. SNAP-25 cleavage by LC / A3V (A1-MLD), a cytosolic LC / A3 chimera that localizes to the plasma membrane independent of intracellular vesicles, was not inhibited by coexpression with of DN Rab27b, DN Rab5, DN Rab3a, or DN Rab4 Fig. QB\650053.01105\92299987.1 Page 87 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 19F-G. Overall, these data are consistent with LC / A1 utilizing the fast synaptic vesicle recycling pathway to stably associate with the plasma membrane for efficient SNAP-25 cleavage.

[0321] Intracellular stability defines LC / A potency and duration of action

[0322] Intracellular eukaryotic proteins typically have an average half-life that ranges from 1.5-48 hours (27–29), yet BoNT / LC persists in motor neurons for weeks to months and continues to cleave SNARE proteins (1, 30). Retention of SNAP-25 cleavage measures BoNT / A duration of 25 action in cultured cells (1), where BoNT / A1 has a longer duration of action than vesicle bound BoNT / A3 (5), and BoNT / A1-A3 chimeras showed LC defined the duration of BoNT action (8). To define the basis for LC / A potency and duration of BoNT action, two novel cell-based assays were developed.

[0323] Measuring the reappearance of uncleaved SNAP-25 to indicate decay of intracellular LC / A in the motor neurons (1), motor neurons transfected with identical moles of mRNA encoding membrane localized LC / A1, vesicle associated LC / A3, and cytosolic LC / A3V, showed a slower reappearance of uncleaved SNAP-25 for LC / A1 and LC / A3 than LC / A3V over 22 days Fig 20A. This implicates that cytosolic LC / A3V was less stable than membrane localized LC / A1 and vesicle associated LC / A3. Motor neuron mRNA transfection experiments with mRNA encoding LC / A1-A3MLD and LC / A3-A1MLD and monitoring SNAP-25 cleavage for 72 days showed that the A subtype of the MLD, which previously was shown to determine membrane localization of LC / A1, plays a significant role in LC stability FIG 20B. This is the first indication that intracellular localization defines LC / A duration of action.

[0324] Next, assessment of intracellular LC / A expression and action in N2A cells provided a mechanism for intracellular LC / A potency of SNAP-25 cleavage in cells. Initial experiments showed that an overnight transfection of N2A cells with two-fold serial dilutions of pEGFPLC / A (A1, A3, or A3V) could be monitored for intracellular SNAP-25 cleavage and LC expression (via GFP signal), enabling dose-dependent intracellular potency measurements Fig 20C. In in vitro assays, earlier studies showed LC / A3 cleaved SNAP-25 at a similar the rate as LC / A1 (31). However, at equivalent moles of transfected pEGFP-LCs intracellular LC / A3 had a ~ 3-fold greater cleavage of SNAP-25 than LC / A1 Fig 20D-E, and an ~30-fold greater cleavage of SNAP-25 than LC / A3(V) Fig.20D-E. At equivalent plasmid transfections, LC / A3V had a statistically lower steady state expression than either LC / A3 or LC / A1. To assess the basis for the lower steady state expression, pEGFP-LC / A3V was used as a platform to synthesize pEGFPLC / A3V (A1 N), pEGFP-LC / A3V (A1 MLD), and pEGFP-LC / A3V (A1 QB\650053.01105\92299987.1 Page 88 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION N, MLD) chimeras. N2A cells transfected with two-fold serial dilutions showed the LC / A3(V)- A1 chimeras possess protein expression levels and potency of SNAP-25 cleavage that correlated with the A1- or A3-source of MLD Fig.20F.

[0325] Assessment of full-length BoNT / A3V showed that composition of the N terminus modulated BoNT potency, with BoNT / A3V possessing an ~ 5-fold lower potency lower Rotarod- and DAS- score than BoNT / A3 in a mouse model of botulism Fig 20G-H. The slightly shortened in vivo duration of BoNT / A3V versus BoNT / A3 indicates a much greater difference at equimolar amounts, as 5 times more total protein of BoNT / A3V had to be injected into mice to see paralysis. Thus, the actual difference in duration is greater than implied by the in vivo studies, and the biological properties of full-length BoNT / A3V correlated with LC / A3V cellular properties. Overall, these studies showed that the N terminus and MLD mediate stable LC / A1 membrane association for the long duration of BoNT / A1 action.

[0326] Discussion

[0327] This is the first study to map LC / A1 intracellular trafficking to the plasma membrane to cleave SNAP-25 and defining the dynamics of LC / A1 intracellular movement via the fast synaptic vesicle signaling pathway. Previous studies showed intracellular LC / A1 localizes stably to the plasma membrane, while LC / A3 localizes predominantly on vesicles and in the cytosol, with dynamic trafficking on and off the plasma membrane, while the LC / A3V variant was primarily present in the cytosol (5). Nocodazole, a microtubule disrupter, displaced vesicle localized LC / A3 to the cytosol of N2A cells but not membrane localized LC / A1 Fig. 15A.

[0328] Therefore, microtubules are required for the dynamic intracellular trafficking of LC / A to the plasma membrane. BoNT entry into neurons is a multistep entry process, where BoNT / A1 binds to a ganglioside / synaptotagmin 1 complex and to SV2, a synaptic vesicle protein, on the surface of a neuron prior to BoNT endocytosis (32, 33). BoNTs enter cells in the lumen of vesicles primarily through clathrin-dependent pathways (34). Studies on BoNT- HC entry into N2A cells showed that treatment with nocodazole decreased HC binding, while genistein, an inhibitor of caveolae-mediated endocytosis and actin networks, did not reduce HC binding (35, 36). Our intracellular trafficking data now complement these studies to show that BoNT / A utilizes microtubules for entry and intracellular trafficking. This data is further strengthened by the observation of bidirectional movement of LC / A1 in neurite sprouts of N2A cells treated with CD (data not shown), which is consistent with a previous study showing that CD treated cultured neurons remain sensitive to BoNT / A cleavage of SNAP-25 (37). QB\650053.01105\92299987.1 Page 89 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0329] Early endosome formation, which is necessary for synaptic vesicle cycling, is mediated by Rab5, which colocalized with LC / A1. Consistently, DN Rab5 inhibited LC / A1 trafficking to the plasma membrane, sequestering LC / A1 on intracellular vesicles Fig. 16A (20). Rab5(a-c) (42) mediates clathrin-dependent endocytosis, a major pathway for SV retrieval from the plasma membrane (11, 34, 43). Previously, Rab5 has been implicated in EGF (44) and Ricin (45) trafficking but not Shiga Toxin endocytosis (44) and in targeting by Yersinia ADPribosyltransferase, a glycosylating toxin from C. difficile (46). In Drosophila, DN Rab5 mediates the homotypic fusion of SVs, increasing SV size and inhibiting neurotransmitter release (47). Furthermore, DN Rab5 inhibits early endosome fusion (48). At steady state, co- expression of DN Ra b5 statistically inhibited LC / A1 membrane localization Fig. 16A and cleavage of membrane localized SNAP-25 Fig. 19D-E, indicating that DN Rab5 blocks a necessary step in the LC / A1 trafficking pathway to the plasma membrane. Inhibition of intracellular trafficking and SNAP-25 cleavage by DN Rab5 supports involvement of early endosomes in LC / A1 trafficking, prior to the formation of Rab4-positive late endosomes, which is also consistent with the cytosolic co-localization of Rab5 and LC / A1 FIG 15B.

[0330] Both, Rab4 and Rab11 facilitate endosomal recycling prior to the formation of SVs (19). As such, kinesin-2-mediated endosome recycling is categorized into two populations where Rab4-mediates fast recycling and Rab11-mediates slow recycling (19, 49, 50). Ricin toxin action has been reported to be sensitive to DN-Rab4 (45). While Rab4 and Rab11 are both found 15 on recycling endosomes, LC / A1 showed a higher degree of colocalization with Rab4 over Rab11 Fig. 15B, and DN Rab4, but not DN Rab11, inhibited LC / A1 trafficking to the plasma membrane Fig.16A. In addition, DN Rab4 was more efficient at inhibiting LC / A1 cleavage of SNAP-25 than DN Rab11 Fig. 19E. This indicates that LC / A1 preferentially utilizes the Rab4-mediated fast synaptic vesicle recycling route to reach the plasma membrane. In neurons, Rab3a and Rab27b control SV docking, fusion, and exocytosis (19). Rab3(ad) (51) is essential for fast kinesin1-mediated anterograde trafficking and vesicle assembly on microtubules (52). While Rab3b, 3c, and 3d are redundant, KO models showed that the removal of Rab3a was lethal post-birth (53). Rab3 and Rab27(a-b) (54) are structurally related, partially co-localized, and share effector proteins such as Rabphilin3a, which binds to Rab3 and preferentially to Rab27 and regulates SV cycling prior to trafficking and localization at the plasma membrane with SNAP-25 (39, 40). A higher colocalization of LC / A1 was observed with Rab27a than Rab3a Fig. 15C and DN Rab3a or DN Rab27b co-expression inhibited LC / A1 trafficking to the plasma membrane Fig. 16A. Inhibition of SNAP-25 cleavage by LC / A1 by DN Rab3a or DN Rab27b Fig.19E is consistent with previous work showing Rab3 QB\650053.01105\92299987.1 Page 90 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION is necessary for SNARE complex assembly and cellular sensitivity to BoNT (55). While present in overlapping pools, temporally Rab3 and Rab27 transition distinct SVs (39), which could explain the inhibition of LC / A1 trafficking to the plasma membrane upon expression of either DN Rab3a or DN Rab27b, yet greater colocalization with Rab27a. Overall, these data demonstrate that late synaptic vesicle cycling is a step in LC / A1 membrane localization and SNAP-25 cleavage. While DN Rab7 did not interfere with LC / A1 trafficking, localization, and cleavage of SNAP-25 Fig. 16A, Fig. 19E at the plasma membrane, Rab7 is necessary for productive intoxication and sorting of the Shiga Toxin B subunit (56). Colocalization of LC / A1 with Rab7 and other Rab GTPases involved in retrograde protein trafficking, such as Rab5 Fig. 15C, may reflect a common interaction between LC / A1 and intracellular vesicles involved in both retrograde and anterograde SV recycling pathways that delivers LC / A1 to the plasma membrane, as seen with LC / A3V (A1 MLD) Fig.19G. This is consistent with a large body of literature supporting retrograde and anterograde trafficking of BoNT / A1 in motor-neurons (5, 6, 9, 57). In the N2A model, which lacks long axonal and neurite connections of motor-neurons, pathways are not expected to be essential for LC / A1 function and membrane localization. However, we propose that in motor-neurons, Rab5 and Rab7 depended axonal transport systems are utilized to distribute LC inside the neuron to reach all synapses. Altogether, these data demonstrate LC / A1 utilizes recycling synaptic vesicles for transport to the plasma membrane.

[0331] In a previous study, we identified the N-terminal 17 aa (N-term) of LC / A1 and an internal ~75 aa motif (MLD) as critical for LC / A1 membrane localization, whereas LC / A3 has an altered 5 MLD that results in vesicle localization, and LC / A3V additionally contains an altered N-term resulting in cytosolic localization (9). Disruption of vesicle association of LC / A3 by nocodazole, a microtubule inhibitor, and the finding that membrane localization of the LC / A3V(A1-MLD) chimera was not affected by DN Rabs of the fast synaptic vesicle cycling pathway, thus implicates the N terminus in vesicle association and intracellular trafficking. Using a novel mRNA cell-based assay to investigate duration of action of intracellular LC, we further demonstrated a role for both the N terminus and the MLD in defining duration of action Fig.20A, B, and using animal studies we showed a role for the N- terminus in determining potency Fig. 20G, H. Altogether, these data confirm a correlation of intracellular localization with potency and duration of action, with contributions by both the LC / A N terminus, which was implicated in intracellular trafficking, and the MLD, previously implicated in stable LC / A1 membrane localization (9). QB\650053.01105\92299987.1 Page 91 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0332] Our findings identify new potential host proteins for BoNT therapy (11), including Rabip4, a Rab4 effector involved in controlling early endosomal trafficking from recycling to sorting endosomes, and Rabphillin 3A (38), a Rab3 / Rab27 effector that binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis (39–41).

[0333] Based on our results combined with data from previous studies on Rab GTPases action (11, 26) and BoNT trafficking and LC / A localization (5, 35, 57), we propose a model of LC / A1 intracellular trafficking Fig. 21. LC / A1 associates with a Rab11 / Rab4-positive recycling endosome, mediated by the N terminal 17 amino acids (5). LC / A1 then preferentially utilizes the fast Rab4-positive recycling endosomes and transitions to Rab3 or Rab27-positive synaptic vesicles after recycling endosomes are primed and loaded with neurotransmitters. When juxtaposed to the plasma membrane, the membrane localization domain of LC / A1 (residues 275-334) binds and aids in cleavage of SNAP-25, presenting LC / A1 as a stably membrane localized protein, where the LC continues to cleave SNAP-25 (9). A necessary step in LC / A1 trafficking is the formation of Rab5 early endosomes which are critical for the formation of downstream vesicular bodies, such as Rab4-positive recycling endosomes. Rab5 and Rab7 are also implicated in protein degradation via phagosomes, thus co-localization with these Rabs may implicate a role for the phagophore / autophagosome in slow LC / A1 degradation.

[0334] Materials and Methods for Example 5

[0335] Reagents were purchased from Life Technologies (Grand Island, NY, USA) unless otherwise specified.

[0336] Engineering 5 of EGFP-LC / A Constructs and Rab Dominant Negative

[0337] Plasmid pEGFP-C3 encoded DNA from A1 (1-450) and A3LM (1-446), were engineered as described previously (5). Enhanced green fluorescent proteins (EGFP) fusions with LC / A1, LC / A3LM, and Rab GTPases3a, 4, 5, 7, 11, and 27a were subcloned into the SacI- BamHI restriction sites of pEGFP-C3. Primers were designed and obtained from New England Biolabs® NEBaseChanger® (Ipswich, MA, USA) to engineer dominant negative (DN) variants of the Rab proteins: Rab3a (T36N), Rab4 (S22N), Rab5 (S34N), Rab7 (T22N), Rab11 (S25N), and Rab27b (T23N).

[0338] Cell Culture

[0339] Neuro-2A (N2A) cells were from ATCC (Manassas, VA, USA) CCL-131. N2A cells were cultured in complete essential media supplemented with 10% fetal bovine serum, 1x penicillin streptomycin, 0.1% sodium bicarbonate, 1mM sodium pyruvate, and 1% nonessential amino acids in humidified 5% CO2 (v / v) at 37 °C. QB\650053.01105\92299987.1 Page 92 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION

[0340] Human motor neurons were cultured and prepared as is known in the art and as described above.

[0341] Transfection and cytoskeleton inhibitor treatment of N2A cells

[0342] N2A cells were transfected as previously described (58). N2A cells were seeded in a 24-well plate at 50,000 cells / well on acid-etched glass coverslips coated with Poly-D lysine (1:500).

[0343] Cytoskeleton inhibitor: The following day, N2A cells were transfected (Lipofectamine LTX; Invitrogen™ (Waltham, MA, USA)) with 500ng of the indicated plasmid, as described earlier (5). Following an overnight transfection, cells were treated with 4μM of Cytochalasin D (C8273, Sigma-Aldrich, St. Louis, USA), an actin inhibitor, or 9μM of nocodazole (M1404, Sigma-Aldrich, St. Louis, USA), a microtubule inhibitor, for 60 min.

[0344] DN Rab GTPase Colocalization: The following day, N2A cells were transfected (Lipofectamine LTX; Invitrogen™ (Waltham, MA, USA)) with 500ng of pEGFP-LC / A1, as described earlier (5).

[0345] DsRed-A1 or A3V (A1 MLD) and EGFP-DN Rab GTPase: The following day, N2A cells were transfected (Lipofectamine LTX; Invitrogen™ (Waltham, MA, USA)) with 2000ng of pDsRed-LC / A1 or pDsRed-LC / A3V (A1 MLD) and 500 ng of pEGFP-DN Rab GTPase the indicated plasmid, as described earlier (5).

[0346] DN Rab GTPase SNAP-25 cleavage: The following day, N2A cells were transfected (Lipofectamine LTX; Invitrogen™ (Waltham, MA, USA)) with 31.25 ng of pEGFP-LC / A1, 125 ng of pEGFP-DN Rab GTPase(s) an pEGFP bringing total ng of plasmid to 656.25, as described earlier (5).

[0347] Immunofluorescent Imaging (IF)

[0348] After an overnight transfection, N2A cells were fixed with 4% (w / v) paraformaldehyde for 15 min at RT. N2A cells were then washed 2x with RT PBS and blocked for 60 min in 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 DBPS (14040182, Gibco™, IL, USA). Unless otherwise specified.

[0349] Cytoskeleton or Rab: N2A cells were incubated with primary antibodies: 1:5000 rabbit α-ß-3 tubulin (ab52623, Abcam, 45 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) shaking, overnight at 4°C in 5% FBS QB\650053.01105\92299987.1 Page 93 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION (v / v), 1% cold-water fish skin gelatin (w / v), 0.1% Triton-X (v / v), and 0.05% Tween-20 (v / v) in DBPS (incubation solution). The following day, N2A cells were washed 3x (five min) with 0.005% Tween-20 (v / v) in DPBS and incubated with Alexa-Fluor-conjugated secondary or phalloidin-Alexa-fluor 568 nm (1:250) (A12380, Life Technologies, WI, USA) in incubation solution for 60 min at RT, washed 2x in 0.005% Tween-20 (v / v) DPBS, and stained with Hoechst (1:10,000 in DPBS, H21492, Invitrogen™, MA, USA) for 5 min. Coverslips were flipped onto microscope slides and cured with 8μL of Prolong™ Gold Antifade Mountant (Invitrogen, MA, USA).

[0350] GFP and DNA colocalization: After an overnight transfection, N2A cells were washed 2x times with 4°C DPBS and stained with wheat germ agglutinin (WGA) (1:1000, W32466, Invitrogen, MA, USA) as a membrane stain for 30 min at 4°C. Following WGA incubation, N2A cells were washed 2x with DPBS and then fixed with 4% (w / v) paraformaldehyde for 15 min at RT, washed 2x with DPBS, and treatment Hoechst (1:10,000) in DPBS, H21492, Invitrogen™, MA, USA) as a nuclear marker for 5 min at RT. Coverslips were flipped onto microscope slides and cured with 8μL of Prolong™ Gold Antifade Mountant (Invitrogen, Waltham, MA, USA).

[0351] SNAP-25 cleavage assay: N2A cells were incubated with primary antibodies: 1:1500 mouse α-SNAP-25 (ABIN236419, Antibodies-Online (Limerick, PA, USA)), shaking, overnight at 4°C in 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) in DBPS (incubation solution). The following day, N2A cells were washed 3x (five min) with 0.005% Tween-20 (v / v) in DPBS and incubated with Alexa-Fluor- conjugated secondary or phalloidin-Alexa-fluor 568 nm (1:1000) (A12380, Life Technologies, WI, USA) in incubation solution for 60 min at RT, washed 2x in 0.005% Tween-20 (v / v) DPBS, and stained with Hoechst (1:10,000 in DPBS, H21492, Invitrogen™, MA, USA) for 5 min. Coverslips were flipped onto microscope slides and cured with 8µL of Prolong™ Gold Antifade Mountant (Invitrogen, MA, USA).

[0352] Cholera Toxin-B subunit and transferrin: After an overnight transfection of DN Rab GTPases (3a, 4, 5, 7, 11, or 27b) N2A cells were washed 2x times with 4°C DPBS and treated with 200 mM Cholera Toxin Subunit B-Alexa Fluor 594 Conjugate (C34777, Thermo, IL, USA) or 200 mM Transferrin- Alexa Fluor 594 Conjugate (T13343 Thermo, IL, USA) for 5 minutes at 37 °C an 5% CO2. N2A cells were then washed 2x with DPBS and then fixed with 4% (w / v) paraformaldehyde for 15 min at RT, washed 2x with DPBS, and treatment Hoechst (1:10,000) in DPBS, H21492, Invitrogen™, MA, USA) as a nuclear marker for 5 min at RT. QB\650053.01105\92299987.1 Page 94 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION Coverslips were flipped onto microscope slides and cured with 8μL of Prolong™ Gold Antifade Mountant (Invitrogen, Waltham, MA, USA).

[0353] Immunofluorescent imaging

[0354] N2A cells were cultured and analyzed as previously described (5, 58).

[0355] Fixed-Cell imaging: N2A cells were live-imaged on a Nikon Eclipse Ti-inverted microscope using a 60x oil-immersion objective (1.4 NA objective) with Eclipse software for data analysis.

[0356] Live-Cell imaging: Seven h post-transfection with LC / A1, as described above, N2A cells were treated and incubated for 60 min with 4μM of cytochalasin D. N2A cells were live- imaged on a Nikon Eclipse Ti-inverted microscope using a 60x oil-immersion objective (1.4 NA objective) with a heated stage at 37°C (Frank E. Fryer A-50) utilizing Eclipse software for data analysis. Images were acquired every 15 seconds for 15 min. The video was compiled utilizing Nikon Elements AR 1.60.0064-bit software (Melville, NY, USA) and Image J (59, 60).

[0357] Western Blot

[0358] EGFP-TRAP®_MA elution was collected in 2x PBS and subjected to SDS-PAGE GEL as previously described (5). Samples were resolved on a 13.5% SDS-PAGE and transferred to an Immobilon-P polyvinylidene difluoride membrane (PVDF) (Millipore, Billerica, MA, USA). PVDF membranes were rehydrated in methanol, washed in reverse osmosis water, stained with Ponceau (P3504, Sigma-Aldrich, St. Louis, USA), and incubated in blocking solution (2% powder milk (w / v) in 0.1% TBST). PVDF membranes were incubated with 1:4000 or 25000 rat α-EGFP-monoclonal IgG (3H9, Chromotek, Planegg, DE), 1:4000 mouse α-SNAP-25 monoclonal IgG (111011, SYSY, Gottingen, DE), shaking, overnight, at 4°C. Bound primary antibodies were recognized as 1:10,000 α-rat or 1:10,000 α-mouse secondary antibodies conjugated with horseradish peroxidase (Life Technologies, MA, USA). Secondary antibodies were visualized using Super Signal™ West Pico PLUS Chemiluminescent Substrate (34578, Thermo, IL, USA) on an Azure C600 Imaging System (Dublin, CA, USA), using a 60-second exposure.

[0359] References for Example 5

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[0420] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. Specifically, any clostridial neurotoxin (or other SNARE cleaving homolog) light chain mRNA or truncated or extended version thereof can be used for therapeutic purposes, and are considered to be within the scope of this invention. Additional mRNA production or delivery methodology can be and is expected to be applied to deliver the mRNA to target cells QB\650053.01105\92299987.1 Page 100 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION in vivo, and the therapeutic use of the mRNA encoding a SNARE cleaving enzyme to treat diseases related to exocytosis is considered to be within the scope of this invention, regardless of the delivery vehicle or mRNA design.

[0421] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0422] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification. QB\650053.01105\92299987.1 Page 101 of 109

Claims

Atty. Dkt. No.650053.01105 PCT APPLICATION CLAIMS We claim:

1. A method, comprising: administering a synthetic mRNA construct for producing a clostridial neurotoxin (BoNT) or SNARE cleaving homologue light chain or a variant thereof to a subject in need thereof.

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

3. The method of claim 2, wherein the BoNT / A1 light chain or variant thereof is the BoNT / A1 light chain variant, wherein the variant comprises 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 an altered duration of BoNT action as compared to wild type BoNT / A1.

4. The method of claim 3, wherein the BoNT / A1 light chain variant comprises the 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 the light chain of BoNT / A3 or a variant thereof.

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

7. The method of claim 6, wherein the BoNT / A3 light chain variant comprises the 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.

8. The method of claim 1, wherein the BoNT or SNARE cleaving homologue light chain or variant thereof is the light chain of BoNT / E or a variant thereof, or the light chain of BoNT / F or a variant thereof, or the LC of BoNT / B or a variant thereof, or the light chain of BoNT / C or a variant thereof, or the light chain of BoNT / D or a variant thereof, or the light QB\650053.01105\92299987.1 Page 102 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION chain of BoNT / G or a variant thereof, or the light chain of BoNT / Wo or a variant thereof, or the light chain of BoNT / Ef or a variant thereof, or the light chain of BoNT / X or a variant thereof, or the light chain of TeNT or a variant thereof, or the light chain of any other member of the larger family of SNARE cleaving toxins.

9. The method claim 8, wherein the BoNT light chain or 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 comprises one or more amino acid substitutions, deletions, or insertions in the respective membrane localization domain (MLD).

11. The method of claim 10, wherein the BoNT / E light chain variant and / or the BoNT / F light chain variant comprises the low homology domain of BoNT / A1, and / or wherein the BoNT / E light chain variant and / or the BoNT / F light chain variant exhibits 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 any of claims 1-11, wherein the synthetic mRNA construct is formulated in 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 a clostridial neurotoxin (BoNT) or SNARE cleaving homologue light chain or a variant thereof.

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

16. The synthetic mRNA construct of claim 15, wherein the BoNT / A1 or SNARE cleaving homologue light chain or variant thereof is the BoNT / A1 light chain variant, wherein the variant comprises 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 as compared to wild type BoNT / A1. QB\650053.01105\92299987.1 Page 103 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 17. The synthetic mRNA construct of claim 16, wherein the BoNT / A1 light chain variant comprises the 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 cleaving homologue light chain or variant thereof is the light chain of BoNT / A3 or variant thereof.

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

20. The synthetic mRNA construct of claim 19, wherein the BoNT / A3 light chain variant comprises the 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 cleaving homologue light chain or variant thereof is the light chain of BoNT / E or a variant thereof, or the light chain of BoNT / F or a variant thereof, or the LC of BoNT / B or a variant thereof, or the light chain of BoNT / C or a variant thereof, or the light chain of BoNT / D or a variant thereof, or the light chain of BoNT / G or a variant thereof, or the light chain of BoNT / Wo or a variant thereof, or the light chain of BoNT / Ef or a variant thereof, or the light chain of BoNT / X or a variant thereof, or the light chain of TeNT or a variant thereof, or the light chain of any other member of the larger family of SNARE cleaving toxins.

22. The synthetic mRNA construct of claim 21, wherein the BoNT light chain or 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 comprises one or more amino acid substitutions, deletions, or insertions in the respective membrane localization domain (MLD).

24. The synthetic mRNA construct of claim 23, wherein the BoNT / E light chain variant and / or the BoNT / F light chain variant comprises the low homology domain of BoNT / A1, and / or wherein the BoNT / E light chain variant and / or the BoNT / F light chain QB\650053.01105\92299987.1 Page 104 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION variant exhibits 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 the synthetic mRNA construct of any of claims 14- 24.

26. The composition of claim 25, wherein the synthetic mRNA construct is formulated in lipid nanoparticles or any suitable delivery vehicle.

27. A BoNT / A3 variant comprising a heavy chain (HC) and a light chain (LC), wherein the LC comprises the 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 the 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 for treating a subject, the method comprising administering a therapeutically effective amount of the synthetic mRNA construct of any of claims 14-24 or the composition of claim 25 and / or 26.

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

33. The synthetic mRNA construct of claim 32, wherein the BoNT LC or variant thereof comprises a variant of BoNT / A1 LC, BoNT / A3 LC, BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G LC.

34. The synthetic mRNA construct of claim 33, wherein the variant of BoNT / A1 LC, BoNT / A3 LC, BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G LC comprises a domain substitution.

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

36. The synthetic mRNA construct of claim 35, wherein the domain substitution of the variant of BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G QB\650053.01105\92299987.1 Page 105 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION LC comprises a substituted N-terminus, a substituted membrane localization domain (MLD), or both.

37. The synthetic mRNA construct of claim 36, wherein the variant of BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G LC comprises 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 / D LC, 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 variant of BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G LC comprises a substituted MLD from BoNT / A1 or BoNT / A3.

40. The synthetic mRNA construct of any one of claims 35-39, wherein a duration of action of the variant of BoNT / B LC, BoNT / C LC, BoNT / D LC, BoNT / E LC, BoNT / F LC, or BoNT / G LC is increased compared to a duration of action of the 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 a duration of action of the variant of BoNT / A1 LC is decreased compared to a duration of action of wild type BoNT / A1 LC.

42. The synthetic mRNA construct of claim 41, wherein the domain substitution in the variant of BoNT A1 LC comprises 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 an N-terminus or a MLD, respectively, from one of 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 a modified potency and / or duration of action as compared to a respective wild type BoNT / B LC, BoNT / D LC, BoNT / F LC, or BoNT / G LC. QB\650053.01105\92299987.1 Page 106 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 45. A therapeutic composition, comprising a clostridial neurotoxin (BoNT) variant, wherein the BoNT variant comprises a domain substitution of an N-terminus, a membrane localization domain (MLD), or both.

46. The therapeutic composition of claim 45, wherein the BoNT variant comprises a variant 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 variant of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G comprises a substituted MLD from BoNT / A1 or BoNT / A3.

51. The therapeutic composition of any one of claims 45-50, wherein a duration of action of the variant of BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G is increased compared to a duration of action of the 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 a duration of action of the variant of BoNT / A1 is decreased compared to a duration of action of wild type BoNT / A1.

53. The therapeutic composition of claim 52, wherein the domain substitution in the variant of BoNT A1 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. QB\650053.01105\92299987.1 Page 107 of 109Atty. Dkt. No.650053.01105 PCT APPLICATION 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 an N-terminus or a MLD, respectively, from 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 a modified potency and / or duration of action as compared to a respective wild type BoNT / B, BoNT / D, BoNT / F, or BoNT / G.

56. A therapeutic composition, comprising the synthetic mRNA construct of any of claims 32-44.

57. The therapeutic composition of claim 56, wherein the synthetic mRNA construct is formulated in lipid nanoparticles or any suitable delivery vehicle.

58. A method for treating a subject, the method comprising administering a therapeutically effective amount of the synthetic mRNA construct of any of claims 32-44, the therapeutic composition of any of claims 45-56, or the therapeutic composition of claim 56 or 57. QB\650053.01105\92299987.1 Page 108 of 109

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