The RNA Landscape of In Vivo-Assembled MS2 Virus-Like Particles as mRNA Carriers Reveals RNA Contamination from Host Viruses

Virus-like particles (VLPs) are attractive systems for packaging and delivering therapeutic RNA molecules in vaccine development, protein replacement therapy, and gene editing. Different VLPs carrying target functional RNA have been biosynthesized and demonstrated for biomedical purposes. However, little attention has been paid to what other types of RNA, besides the target RNA, are encapsulated into VLPs, leading to a lack of knowledge of the landscape of RNA cargoes. In this work, we engineered the widely used MS2 VLPs to encapsulate a model cargo mRNA in yeast, with the packaging efficiency and specificity being quantitatively tuned by the copy number of packaging signals. Transcriptome sequencing of the RNA in the VLPs revealed RNA contamination from the hosts and host viruses. This study highlights the necessity of precise VLP and cargo design and a clear background of production hosts to ensure specificity and safety.