[S8] Symposium 8
Infection control strategies with antibody formulation

[S8-4] Delivery of single-domain antibodies into neurons using a detoxified-chimeric toxin

Shin-Ichiro Miyashita¹, Jie Zhang², Sicai Zhang^2,3, Yoshimasa Sagane¹, Charles Shoemaker⁴, Min Dong^2,3 (¹Dept. of Food Aroma Cosme. Chem., Fac. of Bio-ind., Tokyo NODAI, ²Dept. Urol, Boston Child. Hosp., ³Dept. Surg., Dept. Microbiol., Harvard Med. Sch., ⁴Dept. Infect. Dis. Glo. Health, Cummings Sch. Vet. Med. Tufts Univ.)

Delivering therapeutics into the cytosol of the cell is challenging due to the cell membrane barrier. Bacterial toxins bind to the target cells and efficiently deliver their cargo into the cytosol. Here we took advantage of the ability of the toxins as a vehicle to deliver the therapeutics. Botulinum neurotoxins (BoNTs) specifically target and enter motor neurons, and block neurotransmitter release, resulting cause flaccid muscle paralysis known as botulism. BoNTs has an extremely long half-life within the neuron and induces paralysis that lasts for several weeks to months. Antiserum therapy is used to treat botulism, but they can neutralize only before toxins enter neurons. Our delivery platform is an atoxic chimeric toxin, which fused the receptor-binding domain of BoNT with the translocation domain and the catalytically inactivated catalytic domain of BoNT-like toxin BoNT/X. Single-domain antibody (VHH) against BoNT was fused to the chimeric-toxin and produced by E. coli. In vitro validation, the chimeric toxins delivered the VHH into the neuron and neutralized active BoNT in the cytosol. Furthermore, administration of VHH-chimeric toxin shortens the duration of muscle paralysis in a sub-lethal dose of BoNT injected mice, and rescued mice from lethal doses of BoNT. These results provide novel treatment for botulism and establish a platform to target intracellular proteins.