Engineered tRNAs suppress nonsense mutations in cells and in vivo

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Nonsense mutations cause ~11% of inherited genetic diseases by creating premature termination codons (PTCs).
• Current tRNA-based therapies for nonsense mutations lack optimal efficacy and safety.
• No effective treatments exist for individuals with genetic diseases caused by nonsense mutations.

Purpose of the Study:

• To develop an efficient and safe suppressor tRNA (sup-tRNA) therapy for genetic diseases caused by nonsense mutations.
• To fine-tune native tRNAs into sup-tRNAs by optimizing their sequence for specific amino acid properties.
• To evaluate the efficacy and safety of sup-tRNA therapy in preclinical models.

Main Methods:

• Engineered native tRNAs into sup-tRNAs by fine-tuning their sequence.
• Administered sup-tRNAs via lipid nanoparticles (LNPs) intravenously and intratracheally in mice.
• Assessed protein restoration, readthrough at native stop codons using ribosome profiling, and CFTR function in cell and patient-derived models.

Main Results:

• Lipid nanoparticle-delivered sup-tRNAs restored functional protein production in mice with nonsense mutations.
• No discernible readthrough occurred at endogenous native stop codons, indicating high specificity.
• sup-tRNAs re-established cystic fibrosis transmembrane conductance regulator (CFTR) gene expression and function in relevant models, restoring airway homeostasis.

Conclusions:

• Engineered sup-tRNAs represent a potential therapeutic framework for treating genetic diseases caused by nonsense mutations.
• This tRNA-based gene therapy approach demonstrates a high molecular safety profile and targeted efficacy.
• The findings pave the way for developing novel treatments for a significant unmet medical need.