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Published on: August 3, 2018
Engineered mischarged transfer RNAs for correcting pathogenic missense mutations
Yichen Hou1, Wen Zhang2, Philip T McGilvray3
1Committee on Genomics, Genetics and Systems Biology, University of Chicago, Chicago, IL 60637, USA.
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.
Insights
Researchers developed missense-correcting transfer RNAs (mc-tRNAs) to treat genetic diseases caused by missense mutations. This novel RNA therapy shows promise for correcting these common mutations, offering a new treatment avenue.
Area of Science:
- Molecular Biology
- Genetic Engineering
- Therapeutic Development
Background:
- Missense mutations cause ~50% of human genetic diseases, with limited treatment options.
- Current RNA therapies target nonsense mutations, leaving missense mutations unaddressed.
- Transfer RNA (tRNA) engineering offers a potential strategy for missense mutation correction.
Purpose of the Study:
- To engineer missense-correcting tRNAs (mc-tRNAs) for therapeutic applications.
- To establish a versatile pipeline for mc-tRNA development and validation.
- To demonstrate mc-tRNA potential in treating genetic disorders caused by missense mutations.
Main Methods:
- Developed fluorescent protein reporters to assess missense mutation correction efficiency.
- Engineered mc-tRNAs to correct specific serine and arginine missense mutations.
- Validated mc-tRNA function using mass spectrometry, sequencing, and transcriptomic analysis.
Main Results:
- Successfully engineered mc-tRNAs that corrected missense mutations in reporter systems.
- Confirmed amino acid substitutions and mc-tRNA expression, with minimal transcriptomic changes observed.
- Demonstrated mc-tRNA efficacy in rescuing a pathogenic CAPN3 missense mutant relevant to LGMD2A.
Conclusions:
- Established a versatile pipeline for engineering mc-tRNAs to correct missense mutations.
- Validated mc-tRNAs as a potential therapeutic platform for genetic disorders.
- Highlighted the therapeutic potential of mc-tRNAs for previously untreatable missense mutations.
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