# Developing novel botulism antidotes and a platform for intra-neuronal delivery of nanobodies

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA-IRVINE · 2024 · $263,914

## Abstract

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...

## Key facts

- **NIH application ID:** 10980330
- **Project number:** 1R21AI180532-01A1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA-IRVINE
- **Principal Investigator:** Rongsheng Jin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $263,914
- **Award type:** 1
- **Project period:** 2024-05-20 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10980330

## Citation

> US National Institutes of Health, RePORTER application 10980330, Developing novel botulism antidotes and a platform for intra-neuronal delivery of nanobodies (1R21AI180532-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10980330. Licensed CC0.

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