and G.Q. prevention and treatment of intoxication by botulinum toxin. KEYWORDS: Botulinum neurotoxin, mRNA, prophylaxis, heavy-chain antibody, lipid nanoparticle Introduction Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria of the genus produce seven serotypes of BoNTs (BoNT/A C BoNT/G;1,2 of them, BoNT/A, BoNT/B, BoNT/E, and BoNT/F (rare) are the main causes of botulism in humans.3,4 Botulism is a rare but serious motor nerve-paralyzing disease with rapid onset and high mortality; botulism types are classified into six main categories: food botulism, infant botulism, wound botulism, adult intestinal colonization, iatrogenic botulism, and inhalation botulism.5 BoNT consists of a heavy chain TLX1 (HC; molecular weight??100 kDa) and a light chain (LC; molecular weight??50 kDa). The BoNT-LC is usually BoNTs catalytic domain name composed of a metalloproteinase, which inhibits neurotransmitter release from peripheral nerve endings, thus resulting in botulism. The BoNT-HC contains a receptor-binding domain name (Hc domain name) and a transporter structural domain name (HN domain name), which enable toxin absorption in neurons.6,7 Neutralizing antibodies targeting the Hc domain inhibit the binding of BHc to receptors on nerve ending membranes, thereby neutralizing the toxin and effectively protecting against BoNT/B intoxication. In general, vaccination is the most effective means of preventing botulism; however, current pentavalent vaccines against BoNT/A, BoNT/B, BoNT/C, BoNT/D, and BoNT/E have not been approved for human use because their efficacy and side effects remain unconfirmed.8 Unlike antibody-based passive immunization, vaccines do not TAK-733 swiftly generate protective immunity, which limits their application. Particularly during acute intoxication, antibodies confer a critical advantage to a certain extent.9 Equine anti-botulinum serum is currently the only specific therapy for clinical use in patients who have been intoxicated; however, equine immune serum may cause serum sickness and anaphylactic shock.10,11 This necessitates research on modalities for clinical prevention and treatment of BoNT intoxication. In recent years, mRNAs have been an emerging class of therapeutic drugs for the prevention and treatment of various diseases. mRNA delivery of neutralizing antibody genes has also become an attractive disease prevention strategy. In the early 1990s, exogenous mRNA was reported to direct protein expression in vivo.12 Since then, mRNA technology has emerged as a promising drug platform, showcased by the success of mRNA vaccines which demonstrated its potential and developmental prospects. 13 Another rapidly growing area of mRNA technology is in therapeutic antibodies, now one of the fastest-growing pharmaceutical sectors in the market. Compared with antibody drugs, mRNA-encoded antibodies may have some advantages: simpler production process, higher efficiency, easier emergency preparation ability, low overall and reserve costs, more sustained neutralizing antibody levels, and more long-lasting protective efficacy. Currently, mRNA-encoded antibodies have been applied in the treatment of diseases including viral infections, toxin exposure and malignant tumors.9,14,15 Previous studies reported that formulated RNA encoding VHH heteromultimers confers prevention and therapeutic protection against botulinum intoxication.9,16 These studies highlighted RNA therapy as an established method for rapidly developing and delivering botulinum toxin-neutralizing antibodies. Our previous study has obtained a heavy chain antibody B9-hFc (the fusion protein of camel-derived VHH and Fc region of human antibody), which has BoNT/B-potent neutralizing activity.17 In this study, we designed and synthesized molecules of mRNA encoding B9-hFc, which established rapid and long-lasting serum antibody levels in vivo and demonstrated preventive effects against lethal doses of BoNT/B. This underscored the potential of mRNA-based therapies in combating type B botulism. Our results provide a feasible reference for exploring alternative protein therapies with reduced cost and increased convenience, which is usually significant in enhancing prevention and treatment of type B botulism. Materials and methods mRNA preparation The mRNA sequence, made up of the phage promoter (T7), 5 UTR, ORF, and 3UTR, was designed, synthesized, and cloned into the vector TAK-733 pUC57 to construct a DNA transcription template. The 5UTR comprised a sequence with high ribosome loading from the NCBI Gene Expression Omnibus database (UTR4; original library ID: 317915).18 The 3UTR comprised mRNA sequences derived from mitochondrial 12S rRNA-related sequences and amino-terminal enhancer of split.19 The mRNA molecules were synthesized through transcription in an in vitro cell-free system by using T7 RNA polymerase (Vazyme, DD4202) using the linearized plasmid TAK-733 template obtained through XbaI (NEB, R0145V) digestion. To reduce immunogenicity, we substituted uridine in.