Engineered tRNAs suppress nonsense mutations in cells and in vivo
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ABSTRACT:
PROVIDER: PRJNA846787 | ENA |
REPOSITORIES: ENA
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Project description:Engineered tRNAs suppress nonsense mutations in cells and in vivo
| PRJNA792410 | ENA
Project description:Engineered tRNAs suppress nonsense mutations in cells and in vivo
| PRJNA789542 | ENA
Project description:we introduce a strategy to repurpose sense-codon decoding tRNA into efficient suppressors of the three nonsense mutation-induced PTCs (UGA, UAG and UAA). The suppressor tRNAs restore function of a model and disease-related protein
2023-04-04 | GSE191048 | GEO
Project description:Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.
| S-EPMC10284701 | biostudies-literature
Project description:Comparison of the overall tRNA expression level and detection of the suppressor tRNA
2023-04-04 | GSE192623 | GEO
Project description:Comparison of the overall tRNA expression level and detection of the suppressor tRNA
2023-04-04 | GSE205660 | GEO
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.
2024-01-04 | GSE236623 | GEO
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.
2024-01-04 | GSE236622 | GEO
Project description:Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon (PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.
| S-EPMC10997658 | biostudies-literature
Project description:WenNonsense mutations change a sense codon into a premature termination codon (PTC) in mRNA and account for approximately 18.5% of human inherited retinal diseases (IRDs)-related mutation. Nonsense suppression therapies by small molecular drugs or suppressor tRNAs (sup-tRNAs) can introduce an amino acid at PTC, thereby, promoting the production of full-length proteins. While sup-tRNA-based therapies have shown promising results in cell culture models, challenges remain for in vivo delivery, particularly regarding efficacy, stability, and safety. In this study, we engineered the body sequence of sup-tRNAArg to enhance readthrough efficiency at clinically relevant PTCs in the RPE65 and ABCA4 genes, as well as at PTCs with +4 nucleotide shifts across all four types. By using the self-complementary adeno-associated virus (scAAV), we achieved restoration of RPE65 protein expression in up to 50.2% RPE cells in a mouse model carrying the RPE65-R44X nonsense mutation. Notably, the engineered sup-tRNAArg significantly restored retinal function and visual-guided behavior in mice, with effects lasting for at least 12 weeks. Furthermore, scAAV8.sup-tRNAArg exhibited minimal retinal toxicity and had negligible effects on the activation of unfolded protein response pathways, which is related to readthrough at normal stop codons. Our findings demonstrate the potential of scAAV-delivered sup-tRNAArg as a translatable therapeutic intervention for inherited retinal diseases harboring nonsense mutations.
2025-04-14 | PXD062957 |