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. Author manuscript; available in PMC: 2022 Aug 19.
Published in final edited form as: Nat Neurosci. 2021 Dec 20;25(2):168–179. doi: 10.1038/s41593-021-00973-8

Figure 7. Engineered anthrax toxins deliver molecular cargo into DRG sensory neurons and block pain in vivo.

Figure 7.

(a) Schematic of exogenous cargo delivery into neurons by the PA + LFN system.

(b) (Left) Design of LFN-DTA linking the N terminal domain of LF (LFN) to the A chain of diphtheria toxin (DTA). (Right) Protein synthesis levels in DRG cultures following 6 h treatment with the indicated concentrations of LFN-DTA ± PA (10 nM) (n=3 experiments). Data represent the mean ± s.e.m.

(c) Design of LFN-LC/AC699S linking the N terminal domain of LF (LFN) to a mutated light chain (LC) of type A botulinum neurotoxin (LC/AC699S).

(d-f) DRG cultures were treated with the indicated concentrations of LFN-LC/AC699S ± PA (10 nM) for 24 h and stimulated with 80 mM KCl for 10 min (n=2 wells/condition). Dotted lines connect the means. (d) SNAP-25 cleavage in cell lysates measured by western blot. Estimated molecular weight markers are shown. (e) Percent cleavage was calculated using band intensities with the following formula: cleaved/(intact + cleaved). (f) CGRP release in the supernatant.

(g) Mechanical sensitivity thresholds in SNI mice that received three daily intrathecal injection of vehicle (PBS; n=6 mice), PA only (500 ng; n=6 mice), LFN-LC/AC699S only (200 ng; n=5 mice) or PA + LFN-LC/AC699S (500 ng + 200 ng; n=6 mice). Data represent the mean ± s.e.m.

(h, i) Mice received three daily intrathecal injection of vehicle (PBS) or PA + LFN-LC/AC699S (500 ng + 200 ng) starting on Day 0 (n=5 mice/group) and were monitored by the rotarod test (h) or wire hang test (i). Data represent the mean ± s.e.m.

(j) The effects of BoNT/A, LFN-LC/AC699S alone and PA + LFN-LC/AC699S tested on an ex vivo mouse phrenic nerve hemidiaphragm (mPNHD) preparation (n=3 experiments). Data represent the mean.

Statistical significance was assessed by two-way ANOVA with Sidak’s post hoc test (b) or two-way RM ANOVA with post hoc comparisons (g, h, i). n.s, not significant, ****p<0.0001. For detailed statistical information, see Supplementary Table 2.