Can Next-generation Sequencing accelerate mRNA vaccine development?

Katalin Karikó and Drew Weissman have been granted the 2023 Nobel prize in Physiology or Medicine for their subtle but significant discoveries concerning nucleoside base modifications (Fig1. ), namely replacement of uridine by N1-methylpseudouridine (m1Ψ), which serves as the cornerstone to make mRNA delivery non-immunogenic and to enable the development of mRNA vaccines against COVID-19 with the unprecedented effectiveness and speed, saving millions of lives and ending not only the pandemic of a disease but also the catastrophic health-care burden globally.

Currently, many new mRNA-based therapies have been vigorously developed for a wide variety of diseases, including other infectious pathogens, cancer and auto-immune diseases. In the meanwhile, the concerns about its safety and potential side-effects have been undoubtfully arisen as happened to other new treatments. Though diverse techniques, such as RT-qPCR, capillary gel electrophoresis, IP-RP-HPLC and immunoblotting have been implemented in the quality analysis of mRNA vaccines, those methods are tedious, expensive and lack of sensitivity in detection essential mRNA features. Consequently, a demand on a robust and combinatory analytical method is urgently needed to evaluate mRNA vaccines integrity, efficiency, and more importantly to monitor contaminants and off-target effect to address those safety and specificity concerns. In the last few years, Next-generation sequencing technology has been advanced with a rapid pace on multiple aspects, including improved sequencing throughput capacity, speed, accuracy, significant reduction of cost and long-read development, promoting its various applications in both research and clinical diagnosis. Can next-generation sequencing assist the development and validation of mRNA vaccines and therapies??


An RNA-sequencing study published recently in Nature communication combined and evaluated both short- and long-read sequencing for mRNA vaccine and therapies analysis. The study proposed a streamlined method, VAX-seq workflow (Gunter et al., 2023) followed by an integrated bioinformatic tool called Mana to gain a comprehensive assessment on those mRNA key attributes, including sequence integrity, 3’-end poly(A) tail length and DNA/RNA contaminants (Fig. 2). Moreover, VAX-seq also provides a solution in the detection of off-target RNAs, which are generated during in vitro transcription as truncated, readthrough or anti-sense form of RNAs triggering innate immune response. Both long- and short-read sequencing revealed the similar on- and off-target RNAs percentage.

Figure 2. mRNA vaccine production and VAX-seq workflow. The schematic diagram illustrates the steps during mRNA manufacture (left panel), and the steps during VAX-seq analysis (right panel). This includes laboratory steps of long-read nano-pore sequencing, followed by bioinformatic steps to analyze output data, including the supporting Mana software toolkit. mRNA vaccine quality features that can be analyzed by VAX-seq are indicated (listed in red and green). In the bottom left corner is an IGV plot comparing Oxford Nanopore and Illumina sequencing of a plasmid DNA template. Coverage indicates the number of reads at each nucleotide position while the lower alignments grey bars indicate unique, individual alignments, with colouring indicating their similarity to the reference genome. Source data are provided as a Source data file (from Gunter et al., 2023).

Furthermore, direct RNA nanopore long-sequencing implemented in this study could detect uridine modification, which is required for improving the translation and stability of mRNA vaccines and reducing innate immune response, without the potential bias caused by the reverse transcription and amplification steps in a regular RNA-seq library preparation. However, the misclassification of N1-methylpseudouridine as cytosines has been observed, suggesting that retraining the base-caller is still needed to permit accurate detection of modified nucleosides. Those advantages proposed by this study indicate the application of long and short RNA-seq can serve as a central method to support the large-scale production, quality control and development of new mRNA vaccines and therapies.