We recently reported that fusion of cell-penetrating peptides (CPPs) to botulinum neurotoxin type A (BoNTA) proteins could improve the efficiency of cellular uptake. Here, we describe steps to produce and evaluate CPP-BoNTA fusion proteins. We present procedures for the expression and purification of recombinant CPP-BoNTA using insect-cell-based baculovirus expression vector system and in vitro characterization of purified proteins. We also detail the analysis of cellular uptake in cell culture and examination of the in vivo performance in mice. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022).¹.

Skeletal muscle is the largest metabolic demanding organ in human body. Alterations of skeletal muscle in shape and size significantly affect its biological functions. Botulinum neurotoxin type A1 (BoNT/A1) has been successfully used in clinics to treat masseter, trapezius and gastrocnemius hypertrophy. Here, we used a healthy rat-based skeletal muscle hypertrophy model to evaluate the muscle-reducing activity of recombinant BoNT/A1 (rBoNT/A1) with genetically fused cell-penetrating peptides (CPPs), which was previously reported to increase the cellular uptake of BoNT/A1. Analyses of treated muscle sections using hematoxylin–eosin and immunofluorescence staining showed that both wild-type rBoNT/A1 without modification (WT-rBoNT/A1) and rBoNT/A1 with CPP fusion (CPP-rBoNT/A1) could induce myocomma atrophy and altered gastrocnemius muscle fiber proportions as a result of denervation and reinnervation. Importantly, rBoNT/A1 with the fusion of a specific CPP, zinc finger protein (ZFP), resulted in the highest degree of muscle atrophy and greatest increase in the ratio of type I muscle fibers over type II fibers. An examination of gastrocnemius muscle cells at the subcellular levels using TEM staining revealed swelled mitochondria and diminished mitochondrial crista upon rBoNT/A1 administration. Transcriptomic RNA sequencing (RNA-Seq) analysis followed by RT-qPCR validation showed that rBoNT/A1 treatment also caused changes in mitochondrial biogenesis and mitophagy. Collectively, our results demonstrated that rBoNT/A1 proteins could alleviate skeletal muscle hypertrophy, with associated alterations of mitochondrial homeostasis.