Optimized Synthesis and Translation of RNA Therapeutics

Stanford researchers have developed improved methods for producing mRNAs. Efficient, robust and high fidelity production of mRNAs is critical for obtaining pharmaceutical quality vaccines, viruses and expression constructs, and for eliminating noise due to batch variation. The researchers optimized conditions for efficient mRNA synthesis, m⁷G-capping, and poly(A)-tailing of in vitro transcribed RNA. Such mRNA synthesis can be performed for an individual mRNA but was optimized to be equally well performed for hundreds of different barcoded mRNAs in parallel in one reaction. They also developed a powerful new platform that uses barcoded synthetic mRNA libraries to measure translation efficiency (via polysome profiling) and stability (via time course RNA sequencing). Such synthetic mRNAs carry a unique nucleotide barcode in the 3' UTR that can be used for mRNA identification in large-scale parallel assays based on RNA-seq. This platform allows high-throughput testing of tens of thousands of rationally and/or computationally designed sequences to achieve optimal protein levels. The nucleotide sequence of mRNA is a primary determinant of efficient cellular gene expression; thus, the ability to perform a large-scale analysis of designed mRNA sequences for their ability to be translated efficiently and remain stable inside cells is essential to developing candidates. The new, massively parallel, powerful reporter assay platform is poised to evaluate existing and novel sequence designs as well as inform future rational designs of mRNA-based therapeutics in broader contexts.

This technology is part of a portfolio of innovations aimed at fighting the COVID-19 pandemic.

Related technologies for optimizing RNA-based therapeutics and vaccine design:

Stanford docket S20-205 - Repurposing the SARS-CoV2 5'-UTR for RNA Based Therapeutics
Stanford docket S20-176 - Software for Rapid Mapping of RNA Structure
Stanford docket S20-135 - Translation Enhancer for Gene Regulation
Stanford docket S19-310 - Rational Design of Ultratight RNA Aptamers against Protein Targets
Stanford docket S19-143 - Primerize: Software for Designing Primers for Rapid RNA Synthesis