Abstract Botulinum neurotoxins (BoNTs) with its seven major serotypes, BoNT/A-G, are the causative agents of neuroparalytic disease botulism. BoNT/A and BoNT/B in particular pose the most serious threats to humans because of their wide prevalence, high potency, and persistence. Each BoNT molecule has a modular architecture consisting of a ~50 kDa catalytic light chain (LC) and a ~100 kDa heavy chain (HC). The HC serves as BoNT’s delivery vehicle, which has high specificity for motor neurons and, following its uptake, subsequently translocates the LC into the neuronal cytosol. The LC is the warhead of the toxin, which acts in the cytosol as a sequence-specific protease to cleave soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). This blocks the fusion of synaptic vesicles and prevents the release of acetylcholine at neuromuscular junctions (NMJ), resulting in paralysis of the affected muscles and death by asphyxiation in severe cases. Currently the only available therapeutics for botulism are equine or human antitoxin serum products that can prevent further intoxication by neutralizing the circulating toxins, but these antibodies have no effect on the internalized LC that is mainly responsible for BoNT’s neurotoxic effects. Therefore, there is an urgent need for an effective treatment for post-exposure therapy for botulism patients. Here we strive to develop a platform for intra-neuronal delivery of nanobodies (aka single-domain antibodies such as camelid VHHs) targeting the LC to neutralize the toxin inside the intoxicated neurons and accelerate recovery of the paralyzed muscles. The general principle of our antidote design is to create genetic fusion proteins consisting of three modules: a LC-inhibiting VHH as the payload, the translocation domain of diphtheria toxin as a delivery module for cytosolic translocation, and a targeting module for highly specific neuron targeting. We propose two Specific Aims to explore two different design types. In Aim 1, we will develop antidotes using the receptor-binding domain of BoNT/A (HcA) as the targeting module for intra-neuronal delivery of VHHs that inhibit the LC proteases of BoNT/A and BoNT/B, respectively. HcA binds neuron-specific synaptic vesicle glycoprotein 2 (SV2) and is solely responsible for neuron-targeting of BoNT/A. These fusions proteins will be subjected to comprehensive biochemical and functional characterization to optimize their designs followed by mouse efficacy studies. In Aim 2, we will develop and characterize novel VHHs that bind SV2 to replace HcA as a neuron-targeting domain for the antidotes, which will expand the flexibility of this delivery vehicle and also minimize the immunogenicity concern. We will then test this second type of antidotes for efficacy in animals. Successful completion of this work will result in the identification of novel antidotes for BoNT/A and BoNT/B that can be further developed as therapeutics to treat post-exposure botuli...