Nonsense mutations, which occur when a sense codon in a protein-coding sequence becomes a premature termination codon (PTC), are responsible for approximately 11% of human genetic diseases.

The translational process of mRNA to protein is carried out by tRNA, which recognizes the codon on mRNA by its own anticodon and transports the amino acid corresponding to the codon to the polypeptide chain in ribosome synthesis.

More than 40 years ago, Prof. Yuet Wai Kan pioneered the treatment of β-thalassemia by suppressor-tRNA (sup-tRNA). Sup-tRNA is capable of inducing nonsense mutations in the read-through, and it has most of the same sequence as natural tRNA, but its anticodon recognizes the terminator by base pairing principle and can recognize the stop codons (UAG, UGA, and UAA). Therefore, when PTC is encountered during protein translation, sup-tRNA can introduce the corresponding amino acid into the peptide chain being synthesized, inducing read-through and thus obtaining a functional full-length protein.

However, tRNA therapies based on this principle have not yet entered clinical trials due to the lack of efficacy, inability to reach therapeutic thresholds, and inadequate safety profile.

On May 31, 2023, researchers from the University of Hamburg, Arcturus Therapeutics, and Emory University collaborated to publish a research paper in Nature entitled "Engineered tRNAs suppress nonsense mutations in cells and in vivo". The study successfully suppressed nonsense mutations in vitro and in vivo by LNP-delivered engineered tRNAs, demonstrating that natural tRNAs can be modified and effectively decode clinically important nonsense mutations with a high safety profile.

Suppressive tRNA therapies face challenges because not every natural tRNA can be modified into sup-tRNA by changing its anticodon. This is partly due to the fact that decoding of sense codons is significantly different from decoding of termination codons in eukaryotes. Additionally, PTC activates the mRNA surveillance pathway, leading to degradation of mutated mRNAs. These factors result in sup-tRNA being unable to establish ideal base complementary pairwise binding, leading to ineffective resolution of early termination of translation and mRNA degradation.

As a result, tRNA-based gene therapy has not yet yielded an optimal combination of clinical efficacy and safety, nor is there a treatment for patients with nonsense mutations.

In this latest study, the research team proposes a strategy to alter natural tRNAs into potent sup-tRNAs by individually fine-tuning their sequences to suit the physicochemical properties of the amino acids they carry. Using the functionally conserved features of the natural tRNA and the regulatory sequence outside the anticodon, the team repurposed the bacterial tRNA to fully bind alanine at the stop codon UGA with a codon-anticodon interaction similar to the base complementary pairing structure formed by the decoding of the tRNA by a sense codon.

The research team used a novel LNP system called LUNAR to encapsulate sup-tRNA and construct a safe LNP-sup-tRNA. The LUNAR system is a new LNP delivery system with superior biodegradability and better safety in vivo. By intravenously injecting or inhaling LNP-sup-tRNA, functional proteins with nonsense mutations were restored in mice without significant readout of LNP-sup-tRNA at endogenous natural stop codons.

In addition, the research team also validated it in human cells. Cystic fibrosis (CF) is an inherited exocrine gland disease that primarily affects the gastrointestinal and respiratory systems and is caused by mutations in the CFTR gene. In human patient cells with mutations in the CFTR advance termination codon gene, the optimized sup-tRNA was able to restore its protein expression and function, as well as airway volume homeostasis. Moreover, this process does not require suppression of nonsense mutation-mediated mRNA degradation.

This study provides a new framework for the development of tRNA-based gene therapies that can efficiently suppress the PTC and restore protein expression in vivo with a high safety profile.

Notably, in March 2022, Prof. Guangping Gao and Prof. Dan Wang of the University of Massachusetts published a paper in Nature. This study successfully used recombinant adeno-associated virus for the first time to deliver sup-tRNA to induce protein translation read-through at nonsense mutations and achieved long-term, stable and safe efficacy in a mouse model of mucopolysaccharide storage disorder type I with UAG nonsense mutations.

In recent years, tRNA-based gene therapy is becoming the new rage, and several therapeutic tRNA companies have completed large funding rounds since 2021. These companies are focused or partially focused on tRNA therapeutics, which treat diseases by engineering tRNAs to carry the correct amino acids and introduce them into the position of PTCs, allowing the protein to continue translation.

It is estimated that about 11% of genetic diseases are due to PTCs, so only one such sup-tRNA is theoretically needed to eliminate the premature termination of translation caused by this nonsense mutation and thus cure thousands of different genetic diseases.

These new findings are opening a whole new door for gene therapy.


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