Synthetic Self-Assembling Clostridial Chimera for Modulation of Sensory Functions

Abstract

Clostridial neurotoxins reversibly block neuronal communication for weeks and months. While these proteolytic neurotoxins hold great promise for clinical applications and the investigation of brain function, their paralytic activity at neuromuscular junctions is a stumbling block. To redirect the clostridial activity to neuronal populations other than motor neurons, we used a new self-assembling method to combine the botulinum type A protease with the tetanus binding domain, which natively targets central neurons. The two parts were produced separately and then assembled in a site-specific way using a newly introduced ‘protein stapling’ technology. Atomic force microscopy imaging revealed dumbbell shaped particles which measure ∼23 nm. The stapled chimera inhibited mechanical hypersensitivity in a rat model of inflammatory pain without causing either flaccid or spastic paralysis. Moreover, the synthetic clostridial molecule was able to block neuronal activity in a defined area of visual cortex. Overall, we provide the first evidence that the protein stapling technology allows assembly of distinct proteins yielding new biomedical properties.

Synaptic Vesicle-Recycling Machinery Components as Potential Therapeutic Targets

“…Recombinant atoxic mutants, made by Escherichia coli, can be engineered quickly and efficiently as useful vehicles for delivery to central neurons (Li et al, 2001). Using protein stapling technology, a chimera of botulinum neurotoxin A and the tetanus binding domain was created to specifically target CNS function and spare the neuromuscular junction (Ferrari et al, 2013). By limiting muscle paralysis, this may be useful for the future treatment of epilepsy or chronic pain.…”

Engineering of botulinum neurotoxins as novel therapeutic tools

“…After intrathecal injection of this stapled toxin (100 ng), it inhibited mechanical hypersensitivity in a rat model of CFA-induced inflammatory pain. Interestingly, such a high concentration did not produce any paralysis (flaccid or spastic) [65]. This inventive methodology offers a versatile platform for attaching different ligands to the VAMP component, which display abilities to target various cell types or subpopulation [66].…”

“…It binds to cultured hippocampal neurons, enters, and cleaves SNAP-25 with a potency only threefold less than BoNT/A. Substitution of H C /A with H C /TeTx created another variant [65]. After intrathecal injection of this stapled toxin (100 ng), it inhibited mechanical hypersensitivity in a rat model of CFA-induced inflammatory pain.…”

Uptake and transport of clostridial neurotoxins

“…Retargeting of CNTs to different neuronal populations other than MNs was achieved by a new self-assembling method, which enables the stapling of the BoNT/A protease with H C T [183]. Both recombinant proteins were produced separately and assembled using protein stapling technology, which exploits the high-affinity interaction of paired SNARE motifs [183].…”

“…Both recombinant proteins were produced separately and assembled using protein stapling technology, which exploits the high-affinity interaction of paired SNARE motifs [183]. Mechanical hypersensitivity was observed as a result of introducing the stapled chimera into a rat model of inflammatory pain, and neuronal activity was blocked in a specific area of the visual cortex [183].…”