New botulinum neurotoxin constructs for treatment of chronic pain

Abstract

Chronic pain affects one in five people across human societies, with few therapeutic options available. Botulinum neurotoxin (BoNT) can provide long-lasting pain relief by inhibiting local release of neuropeptides and neurotransmitters, but its highly paralytic nature has limited its analgesic potential. Recent advances in protein engineering have raised the possibility of synthesising non-paralysing botulinum molecules for translation to pain sufferers. However, the synthesis of these molecules, via several synthetic steps, has been challenging. Here, we describe a simple platform for safe production of botulinum molecules for treating nerve injury-induced pain. We produced two versions of isopeptide-bonded BoNT from separate botulinum parts using an isopeptide bonding system. Although both molecules cleaved their natural substrate, SNAP25, in sensory neurons, the structurally elongated iBoNT did not cause motor deficit in rats. We show that the non-paralytic elongated iBoNT targets specific cutaneous nerve fibres and provides sustained pain relief in a rat nerve injury model. Our results demonstrate that novel botulinum molecules can be produced in a simple and safe manner and be useful for treating neuropathic pain.

Figure 1.. Production of isopeptide-bonded BoNT molecules.

(A) Schematic of the structural organisation of BoNT/A (upper panel) with additions of the SpyCatcher elements to two botulinum parts (lower panel). (B) Coomassie-stained SDS–PAGE showing the purified proteins and spontaneous assembly of isopeptide-bonded BoNT. (C) Coomassie-stained SDS–PAGE showing isolation of iBoNT from the unreacted excess of SpyTag–HC by gel filtration. (D) Structural models of native BoNT/A (left), non-elongated iBoNT (centre), and elongated iBoNT (right) molecules based on known crystal structures (Lacy et al, 1998; Lerman et al, 2000; Li et al, 2014). (E) Coomassie-stained SDS–PAGE demonstrating increasing molecular weights of iBoNT molecules compared with the native BoNT/A molecule, in non-reducing (left) and reducing conditions (right). The light chains (asterisks) become separated from the heavy chains (arrowheads) upon reduction in the critical disulphide bond.

Figure S1.. Dimensions of the stapled Bitox and el-iBoNT may compromise their function specifically in small synaptic vesicles but not in large peptidergic vesicles.

(A) Comparison of the lengths of the previous Bitox molecule (left) with the new el-iBoNT molecule (right). (B) Lengths of native BoNT and el-iBoNT molecule in relation to a small synaptic vesicle (left) and a peptidergic vesicle (right). Structures used: BoNT/A (light chain: blue, translocation domain: orange, HC domain: red) from PDB 3BTA (Lacy et al, 1998); SNARE helix (light blue, top left) from PDB 1SFC (Sutton et al, 1998); syntaxin extension sequence (light blue) from PDB 1EZ3 (Lerman et al, 2000); Spycatcher–Spytag (green) from PDB 4MLI (Li et al, 2014); and SV2C (luminal domain: magenta, translocation domain: grey) from PDB 4JRA and modelling (Benoit et al, 2014).

Figure S2.. Formation and purification of el-iBoNT and its component parts.

(A) Coomassie-stained SDS–PAGE showing the purified proteins and spontaneous assembly of el-iBoNT within 2 h of mixing. (B) Coomassie-stained SDS–PAGE showing separation of el-iBoNT from the unreacted excess of Spytag–HC by gel filtration. Fraction 8 was used in all experiments. Source data are available for this figure.

Figure 2.. Functional testing of botulinum molecules.

(A) Immunoblot (left) and graph (right) showing SNAP25-cleaving activity of the native BoNT/A and two novel botulinum molecules. Cleaved SNAP25 (cSNAP25) migrates slightly faster than native SNAP25. Rat DRG neurones were treated with botulinum molecules at indicated concentrations for 68 h before performing immunoblotting using an anti-SNAP25 antibody with an anti-syntaxin antibody serving as a loading control. The proportion of SNAP25 that had been cleaved was determined by band densitometry (n = 3, two-way ANOVA with Tukey’s multiple comparisons test). (B) Schematic showing detection of botulinum-cleaved SNAP25 by cSNAP25 antibody (left). Immunocytochemical images showing a similar degree of cleavage of SNAP25 (red) by el-iBoNT and native BoNT in rat DRG neuronal cultures. The DRG neurons were treated with either BoNT/A or el-iBoNT at 1 nM concentration, for 24 h, followed by immunostaining for beta-III tubulin (green) and cSNAP25 (red). Scale bars: 100 μm. (C) Graph showing changes in compound muscle action potentials recorded in rat gastrocnemius muscle after subcutaneous injections of two iBoNT molecules (10 ng). The electromyography data obtained 72 h post-injection show that el-iBoNT elicits significantly less motor deficit compared with non-elongated iBoNT (P < 0.001, n = 4, two-way ANOVA, Tukey’s post hoc test). (D) Immunohistochemical examination of cleaved SNAP25 (cSNAP25, red) in rat gastrocnemius muscle after subcutaneous injections of the two iBoNT molecules (10 ng) reveals reduced cleavage of SNAP25 at neuromuscular junctions (NMJs) in the case of elongated iBoNT (scale bar: 50 μm). Bungarotoxin staining (green) delineates NMJs. The bar chart shows the percentage of NMJs carrying cleaved SNAP25. The percentage of NMJs with cSNAP25 is significantly reduced in animals injected with el-iBoNT compared with non-elongated iBoNT (P < 0.01, n = 4, one-way ANOVA, Tukey’s post hoc test).

Figure S3.. Immunoblot showing that the individual LHN–Spycatcher and LHN–syx–Spycatcher components do not cleave SNAP25 in the absence of the binding domain.

Rat DRG neurones were treated with individual botulinum components at indicated concentrations for 68 h before performing immunoblotting using an anti-SNAP25 antibody.

Figure S4.. iBoNT paralyses rat leg after injections into the left gastrocnemius muscle (black arrows), whereas el-iBoNT or its component parts do not.

(A) Image of a rat injected with 20 ng of iBoNT is shown. The injected leg was splayed and unable to support weight after 48 h. (B, C, D) Images of rats injected with 20 ng of el-iBoNT (B) or 100 ng of individual botulinum parts (C, D) demonstrate the lack of muscle paralysis. (E) Bar chart showing the number of rats visibly affected by the injections of native 1 ng BoNT or 20 ng el-iBoNT into the gastrocnemius muscle (n = 3).

Figure 3.. Dose-dependent effects of intraplantar injections of el-iBoNT in the rat spared nerve injury (SNI) model.

Basal paw withdrawal thresholds were measured at day 0, and SNI was performed on day 1. The sciatic nerve was exposed but not manipulated to act as a sham control (grey). On day 5, a single dose of el-iBoNT at indicated amounts or a saline vehicle was administered by intraplantar injection. (A) Time course of mechanical hypersensitivity was assessed by recording paw withdrawal thresholds using the von Frey hair stimulation (left). Robust mechanical hypersensitivity was observed in all animals after SNI surgery in comparison with the sham controls. Injections of el-iBoNT reversed mechanical hypersensitivity of the paw because of nerve injury. Data show log of the mean of the 50% threshold ± SEM. The bar chart shows that at day 28, even the 5-ng group exhibited mechanical sensitivity comparable to sham-operated animals, in contrast to the vehicle-only group (two-way ANOVA with Tukey’s multiple comparisons test, **P < 0.01, n = 4) (right). (B) Immunostaining for Iba-1 reveals microglial activation in rat spinal cord two weeks after SNI injury (central panel), but not in the sham group (left panel). After a single intraplantar injection of el-iBoNT (20 ng), the number of activated spinal microglia, one week after nerve injury, is significantly reduced (right panel and bar chart, right panel, unpaired t test, *P = 0.016) (scale bar: 50 μm).

Figure S5.. Changes in animal behaviour and gait were observed after spared nerve injury and subsequent subplantar injection of either vehicle or el-iBoNT.

Animals injected with vehicle showed reluctance to place ipsilateral hind paw onto the mesh surface and showed reduced weight bearing on the affected limb (left panel, n = 6). In contrast, animals injected with el-iBoNT more readily made contact with the mesh flooring and showed more normal gait behaviour (right panel, n = 6). Images were taken 7 d after spared nerve injury and 3 d after subplantar injections.

Figure S6.. Imaging of botulinum-cleaved SNAP25 in rat nerve tissues following intraplantar injections.

(A) Immunohistochemical image showing the absence of cleaved SNAP25 (red, background) in ipsilateral L5 dorsal root ganglion after intradermal paw injection of el-iBoNT in naïve rat. The image was taken 7 d post-injection. Scale bar: 40 μm. (B) Immunohistochemical image showing the absence of cleaved SNAP25 (red, background) in L5 spinal cord area after intradermal injection of el-iBoNT. Staining for cSNAP25 was not observed in either the ventral or dorsal horns of ipsilateral transverse spinal cord sections. Scale bar: 200 μm. (C, D) Higher power images of the bundle of nerve fibres within the dermis from Fig 4A with evidence of colocalisation (yellow) of cleaved SNAP25 (red) and TRPV1 (green).

Figure 4.. Localisation of cleaved SNAP25 (cSNAP25) in cutaneous tissue injected with el-iBoNT.

Colocalisation of cSNAP25 and TRPV1 in dermal fibres of rat cutaneous tissue. (A) TRPV1 (green) staining seen to coexist (yellow) with cSNAP25 (red) in dermal but not epidermal tissue. Dermal–epidermal boundary shown with a dotted line. Examples of costaining indicated by arrows. (B) NF200, a marker for larger A fibres (green), colocalises with some cSNAP25-positive fibres (red). (C) Dermal arteriole costained with tyrosine hydroxylase (TH, green) and cSNAP25 (red) showing a robust colocalisation of the two markers in sympathetic nerve fibres. Scale bars: a, e, f = 50 μm, and b, c, d = 10 μm.

Figure S7.. Immunoblots showing a direct comparison of SNAP25-cleaving activity of el-iBoNT in rat DRG neurons (top) and differentiated SiMa neuroblastoma cells (bottom) with syntaxin used as a loading control (n = 4).