Engineered tRNAs suppress nonsense mutations in cells and in vivo
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ABSTRACT:
PROVIDER: PRJNA792410 | ENA |
REPOSITORIES: ENA
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Project description:Engineered tRNAs suppress nonsense mutations in cells and in vivo
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Project description:Suppressor transfer RNAs (sup-tRNAs) can rescue disease-causing nonsense mutations by promoting readthrough of premature termination codons (PTCs). Their clinical translation is limited by suboptimal activity and inefficient in vivo delivery. In this work, we combined site-specific chemical modification of sup-tRNAs with cargo-tailored pulmonary lipid nanoparticle (LNP) engineering to overcome these barriers. Incorporation of N1-methyladenosine in sup-tRNAs improved PTC readthrough, enhanced tRNA aminoacylation, prolonged functional persistence, and reduced innate immune activation. High-throughput ionizable lipid screening and formulation optimization identified a sup-tRNA–tailored LNP that efficiently delivered chemically modified sup-tRNAs to the lung. This approach restored cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in bronchial epithelial cells, mouse models, and patient-derived organoids. Thus, LNP-delivered, chemically engineered sup-tRNAs represent a potential therapeutic platform for treating nonsense mutations.
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Project description:Suppressor transfer RNAs (sup-tRNAs) can rescue disease-causing nonsense mutations by promoting readthrough of premature termination codons (PTCs). Their clinical translation is limited by suboptimal activity and inefficient in vivo delivery. In this work, we combined site-specific chemical modification of sup-tRNAs with cargo-tailored pulmonary lipid nanoparticle (LNP) engineering to overcome these barriers. Incorporation of N1-methyladenosine in sup-tRNAs improved PTC readthrough, enhanced tRNA aminoacylation, prolonged functional persistence, and reduced innate immune activation. High-throughput ionizable lipid screening and formulation optimization identified a sup-tRNA–tailored LNP that efficiently delivered chemically modified sup-tRNAs to the lung. This approach restored cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in bronchial epithelial cells, mouse models, and patient-derived organoids. Thus, LNP-delivered, chemically engineered sup-tRNAs represent a potential therapeutic platform for treating nonsense mutations.
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Project description:Missense mutations account for nearly 50% of pathogenic mutations in human genetic diseases, most lack effective treatments. Gene therapies, CRISPR-based gene editing, and RNA therapies including transfer RNA (tRNA) modalities are common strategies for potential treatments of genetic diseases. However, reported tRNA therapies are for nonsense mutations, how tRNAs can be engineered to correct missense mutations have not been explored. Here, we describe missense correcting tRNAs (mc-tRNAs) as a potential therapeutic modality for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs that are charged with one amino acid and read codons of another amino acid in translation in human cells. We first developed a series of fluorescence protein (FP)-based reporters that indicate successful correction of missense mutations via restoration of fluorescence signals. We engineered mc-tRNAs that effectively corrected Serine and Arginine missense mutations in the reporters and confirmed the amino acid substitution by protein mass spectrometry and mc-tRNA expression by tRNA sequencing. We examined the transcriptome response to the expression of mc-tRNAs and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an Arg-tRNAGln(CUG) mc-tRNA to rescue the autolytic activity of a pathogenic CAPN3 Arg-to-Gln mutant involved in limb-girdle muscular dystrophy type 2A. 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.
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Project description:Missense mutations account for nearly 50% of pathogenic mutations in human genetic diseases, most lack effective treatments. Gene therapies, CRISPR-based gene editing, and RNA therapies including transfer RNA (tRNA) modalities are common strategies for potential treatments of genetic diseases. However, reported tRNA therapies are for nonsense mutations, how tRNAs can be engineered to correct missense mutations have not been explored. Here, we describe missense correcting tRNAs (mc-tRNAs) as a potential therapeutic modality for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs that are charged with one amino acid and read codons of another amino acid in translation in human cells. We first developed a series of fluorescence protein (FP)-based reporters that indicate successful correction of missense mutations via restoration of fluorescence signals. We engineered mc-tRNAs that effectively corrected Serine and Arginine missense mutations in the reporters and confirmed the amino acid substitution by protein mass spectrometry and mc-tRNA expression by tRNA sequencing. We examined the transcriptome response to the expression of mc-tRNAs and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an Arg-tRNAGln(CUG) mc-tRNA to rescue the autolytic activity of a pathogenic CAPN3 Arg-to-Gln mutant involved in limb-girdle muscular dystrophy type 2A. 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.
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