Helen Gunter from the Australian Institute for Bioengineering and Nanotechnology at the University of Queensland in Australia spoke at London Calling 2023 about “Using nanopore sequencing to understand the manufacture, delivery, and action of mRNA vaccines.” Gunter explained how they use Oxford Nanopore Technologies devices and sequencing in vaccine development. They spoke about the mRNA ecosystem with mRNA vaccines, siRNA therapeutics, RNA switches in synthetic biology, and dsRNA vaccines for agriculture. The structure of a synthetic mRNA has a 5′ cap, 5′ UTR, coding region, 3′ UTR, and poly A tail. Gunter explained that they are synthesized in vitro. Their lab is collaborating with the BASE facility in the University of Queensland to manufacture vaccines. Synthetic mRNAs are manufactured from a plasmid with a T7 promoter and in vitro transcribed. The mRNA is purified and encapsulated into liquid nanoparticles. Gunter emphasized that they wanted to streamline the mRNA quality assessments. The VAX-seq approach Gunter described is a “streamlined mRNA quality analysis using ONT sequencing” that utilizes several ONT kits. Plasmid DNA templates are sequenced using ligation or rapid sequencing kits. cDNA sequencing of mRNA vaccines helps confirm sequence, identify cryptic promoters, and find run-on transcription issues caused by incomplete plasmid linearization. VAX-seq also provides information about polyA tail and amount of contamination from Escherichia coli from plasmid propagation. Routine monitoring of vaccine production in the BASE facilities is using VAX-seq for COVID-19 mRNA vaccine template sequencing. In an example, they identified an issues with a polyA tail due to a deletion. VAX-seq was also utilized at BASE to sequence mRNA and check mRNA integrity. mRNAs were sequenced with the direct RNA sequencing, identifying minor alteration of mRNA fragment sizes. Gunter emphasized that this approach they developed can utilize Nanopore sequencing tor quality control and improvement of vaccine production practices at BASE. I appreciate the variety of applications and uses of Nanopore devices and sequencing with VAX-seq.
