Engineered tRNAs suppress nonsense mutations in cells and in vivo
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PROVIDER: PRJNA846787 | ENA |
REPOSITORIES: ENA
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Project description:Engineered tRNAs suppress nonsense mutations in cells and in vivo
| PRJNA789542 | ENA
Project description:Engineered tRNAs suppress nonsense mutations in cells and in vivo
| PRJNA792410 | 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:A large number of genetic diseases are caused by various mutations in specific disease genes. A significant proportion (~15%) of these mutations are nonsense mutations that create a premature termination codon (PTC). Consequently, the Nonsense-mediated mRNA Decay (NMD) surveillance pathway degrades a large fraction of PTC-containing mRNA. Translation of the remaining un-degraded PTC-containing mRNA terminates at the PTC, leading to no full-length protein production and hence disease. Therefore, suppressing NMD and translation termination at PTCs becomes an attractive strategy for combating these diseases. This work presents a novel approach, namely targeted PTC pseudouridylation, to suppress nonsense mutations in human cells. By co-transfecting human cells with a PTC-reporter gene and a designer box H/ACA guide RNA gene targeting the PTC, we show that targeted pseudouridylation suppresses both NMD and translation termination at PTCs in the contexts of various disease gene sequences. Furthermore, targeted pseudouridylation exhibits a level of suppression comparable to that of antibiotic treatments, and the suppressive effect is long-lasting in the cell. When targeted pseudouridylation is combined with the antibiotic treatment, a much higher level of suppression is observed. Remarkably, by transfecting a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene alone targeting the PTC, we also observe nonsense suppression, suggesting the high potential of this novel approach in treating PTC-associated diseases. Finally, one of the restored full-length proteins is further tested to be functional. Targeted pseudouridylation appears to be the first RNA-directed gene-specific therapeutic approach that suppresses NMD and concurrently promotes PTC read-through.
2022-12-31 | GSE221516 | GEO