Research ArticleHEALTH AND MEDICINE

Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models

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Science Advances  16 Jan 2019:
Vol. 5, no. 1, eaau7196
DOI: 10.1126/sciadv.aau7196
  • Fig. 1 Engineered rBoNT/B1 toxins inhibit neurotransmitter release with higher potency than rBoNT/B1.

    (A) Inhibition of [3H]-glycine release from primary rat SCNs by rBoNT/B1, rBoNT/B1MY, or rBoNT/B1QW. The logarithmic concentration of each toxin required for IC50 (pIC50) of [3H]-glycine release was 11.46 ± 0.13 (n = 4), 11.96 ± 0.05 (n = 3), and 11.98 ± 0.12 (n = 3) for rBoNT/B1, rBoNT/B1MY, and rBoNT/B1QW, respectively. *P < 0.05, Tukey’s multiple comparison. Maximum achieved mean inhibition of the release was 82.4, 85.1, and 85.2% for rBoNT/B1, rBoNT/B1MY, and rBoNT/B1QW, respectively. (B) Inhibition of [3H]-GABA release from human iCell GABANeurons by rBoNT/A1, rBoNT/B1, rBoNT/B1MY, or rBoNT/B1QW. The pIC50 of [3H]-GABA release was 11.63 ± 0.05 (n = 5), 10.97 ± 0.08 (n = 4), 12.57 ± 0.16 (n = 3), and 12.53 ± 0.11 (n = 3) for rBoNT/A1, rBoNT/B1, rBoNT/B1MY, and rBoNT/B1QW, respectively. ***P < 0.0001, analysis of variance (ANOVA) followed by Tukey’s multiple comparison. Maximum achieved mean inhibition of the release was 91.4, 94.1, 89.5, and 70.8% for rBoNT/B1, rBoNT/B1MY, rBoNT/B1QW, and rBoNT/A1, respectively.

  • Fig. 2 Characterization of a transgenic mouse strain in which the luminal domain of Syt2 is replaced by the human sequence.

    (A) Schematic representation of Syt2 (left) and sequence alignment of the human and murine vesicle luminal domains (right). Non-identical residues are highlighted in red, including F at position 54 of the murine sequence (red underlined). Transgenic mice (TG) incorporate the entire human luminal domain, as shown. (B) Breeding data of WT and various genotypes of transgenic mice. All tested breeding groups produced offspring; however, the productivity index (defined as number of pups per female per week) for groups where the male animals had at least one transgenic allele (tg) was lower than WT mice, which always produced offspring. These results are consistent with a dominant partial male infertility of the modified Syt2. N/A, not assessed. (C) Transgenic mice homozygotes show a normal BW to age profile compared to WT homozygotes (3 to 41 animals per data point). (D) Immunohistochemistry data showing that the expression pattern of Syt1 and Syt2 in brain and spinal cord of transgenic animals was not different as compared to homozygotic WT animals. (E) The expression levels of Syt1 and Syt2 in the cerebellum (CB), cortex (CTX), and hippocampus (HC) of homozygotic transgenic animals was assessed by Western blot analysis. Values are normalized to expression of SNAP-25 in each preparation. There was no significant difference for any of the brain regions or Syt isoforms between transgenic mice and WT animals (P > 0.05, paired t test).

  • Fig. 3 Engineered rBoNT/B1, but not rBoNT/B1, toxins induce rapid hemidiaphragm paralysis in hSyt2 mice.

    (A to C) Contractile force of the hemidiaphragm muscle plotted over time from WT or hSyt2 mice. The phrenic nerve was stimulated at a frequency of 1 Hz with a 20-μs pulse duration. Following tissue stabilization, a toxin was added (blue arrows) to the bath at a final concentration of 10 pM. Whereas rBoNT/B1 induced a rapid paralysis of the muscle from WT mice, paralysis was significantly attenuated (P < 0.0001, unpaired t test) in tissue prepared from hSyt2 mice (A). In contrast, both rBoNT/B1MY (B) and rBoNT/B1QW (C) induced a rapid paralysis in hemidiaphragm preparations from both WT and hSyt2 mice. (D) All data were normalized to the contractile strength before toxin addition and fitted to a four-parameter equation to determine t50. Unpaired t test was used to compare datasets between WT and hSyt2 mice for each toxin. Datasets for all toxins per WT or hSyt2 mice were compared with ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05, ***P < 0.0001. All other comparisons were not significant.

  • Fig. 4 Engineered rBoNT/B1, as well as rBoNT/B1, toxins induce rapid bladder detrusor muscle paralysis in hSyt2 mice.

    (A and B) Contractile force of bladder detrusor muscle strips plotted over time from WT or hSyt2 mice. Bladder detrusor muscles were electrically stimulated with 0.3-ms pulses at 10 Hz to determine the baseline contractile force. Following tissue stabilization, a toxin was added (blue arrows) to the bath at 1 nM final concentration. Both rBoNT/B1 (A) and rBoNT/B1MY (B) induced a comparable paralysis in bladder strip preparations from WT and hSyt2 mice. (C) All data were normalized to the contractile strength before toxin addition and fitted to a four-parameter equation to determine t50. Between tissue types, there was no significant difference for any of the four toxins tested (unpaired t test). Datasets for all toxins per WT or hSyt2 mice were compared with ANOVA, followed by Tukey’s test for multiple comparisons. ***P < 0.0001. All other comparisons were not significant. (D) IHC analysis of urothelium tissue shows low, consistent expression of Syt1 but fails to detect Syt2 in urothelium from WT and hSyt2 mice (inset: positive antibody control from spinal cord tissue).

  • Fig. 5 Engineered rBoNT/B1 toxins have in vivo activity similar to rBoNT/A1.

    (A) rBoNT/B (left), rBoNT/B1MY (middle), and rBoNT/B1QW (right) were injected into the gastrocnemius-soleus muscle complex of the hind paw of WT or transgenic hSyt2 mice. DAS was scored daily during the first 4 days after injection, and the highest average DAS score per dose group during this period is plotted and fitted. ED50 values and lower and upper 95% confidence intervals derived from the fit are given in table S2. In the transgenic mice, rBoNT/B1 had dramatically reduced activity as compared to the WT animal. A high activity also in the transgenic animals was restored with both engineered toxins. (B) Muscle force generated in the hind leg of anesthetized New Zealand white rabbits after injection of rBoNT/A1 or rBoNT/B1MY into the gastrocnemius-soleus muscle complex of the hind paw. The muscle was stimulated at the tibial nerve with pulses of 40 V (50 μs at 0.5 Hz). The muscle force generated by the triceps surae group was recorded for at least 10 consecutive stimulations, and the maximum amplitude of the muscle force generated was calculated for each stimulation. Measurement was done for the side injected with the toxin [ipsilateral (Ipsi)], as well as the noninjected contralateral (Contra) side. Three doses were tested as indicated, and the muscle force was measured once weekly for 5 weeks.

  • Fig. 6 Syt recognition by BoNT/B1 variants.

    (A) Crystal structure of rBoNT/BMY (blue) in complex with hSyt1 (yellow). The three functional domains of BoNT/B1 are labeled, with the catalytic light chain (LC), the translocation domain (HN), and the binding domain (HC). (B) Superposition of the crystal structures of Hc/B (gray) with hSyt1 (yellow) and rSyt2 [red; Protein Data Bank (PDB) code: 2 nm1]. Structures were aligned using the binding domain only. Residues E1191 and S1199 are highlighted in blue; the N and C termini of Syt are indicated. (C) Superposition of the crystal structures of the Hc/B (gray)–hSyt1 (green) complex with rBoNT/BMY (blue)–hSyt1 (yellow). Movement of loop 1197-1201 and the difference in the position of the peptides’ N termini are indicated with blue and black arrows, respectively. (D) Superposition of the crystal structures of rBoNT/BMY (blue) with hSyt1 (yellow) and hSyt2 (red). The shift between the two peptide positions is indicated with a black arrow.

Additional Files

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Longitudinal study of SNARE protein expression in iCell GABANeurons.
    • Fig. S2. Evaluation of Syt2 sequences with L54 in BoNT/B binding site.
    • Fig. S3. Molecular mechanisms of Syt recognition by BoNT/B1 variants.
    • Fig. S4. Electron density maps of the bound peptides and surface electrostatic potential of toxins.
    • Fig. S5. Effect of C-terminal histidine tag on rBoNT/B1 activity.
    • Fig. S6. Production of recombinant BoNTs.
    • Table S1. Dissociation constants (in micromolar) of rBoNT/B1 binding domains for human (h) and rodent (r) GST-tagged Syt1 and Syt2, measured by biolayer interferometry.
    • Table S2. Efficacy and safety of toxins in murine in vivo studies.
    • Table S3. X-ray crystallography: Data collection and refinement statistics.
    • Table S4. Antibodies used in immunohistochemistry studies.

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