Engineered mischarged transfer RNAs for correcting pathogenic missense mutations

Abstract

Missense mutations account for approximately 50% of pathogenic mutations in human genetic diseases, and most lack effective treatments. Gene therapies, gene editing, and RNA therapies, including transfer RNA (tRNA) modalities, are common strategies for genetic disease treatments. However, reported tRNA therapies are for nonsense mutations only. It has not been explored how tRNAs can be engineered to correct missense mutations. Here, we describe missense-correcting tRNAs (mc-tRNAs) as a potential therapeutic for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs charged with one amino acid, but read codons of another in translation. We first developed a series of fluorescent protein-based reporters that indicate the successful correction of missense mutations via restoration of fluorescence. We engineered mc-tRNAs that effectively corrected serine and arginine missense mutations in the reporters and confirmed the amino acid substitution by mass spectrometry and mc-tRNA expression by sequencing. We examined the transcriptome response to mc-tRNA expression and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an mc-tRNA to rescue a pathogenic CAPN3 Arg-to-Gln mutant involved in LGMD2A. These results establish a versatile pipeline for mc-tRNA engineering and demonstrate the potential of mc-tRNA as an alternative therapeutic platform for the treatment of genetic disorders.