Engineered transfer RNAs for suppression of premature termination codons

Abstract

Premature termination codons (PTCs) are responsible for 10–15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. Here we present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identify ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within cystic fibrosis transmembrane conductance regulator (CFTR).

Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides

“…Moreover, a therapeutic drawback of AGs, and more generally for all compounds that stimulate miscoding, are the general inhibitory loss of function effects on both translation elongation and termination. Targeting translation termination codons through the use of suppressor tRNAs (Lueck et al, 2019) presents an alternative approach to aminoglycosides with potential for fewer off-target effects as suppressor tRNAs should only impact the fidelity of stop codon recognition for roughly one third of stop codons, but not the overall fidelity of translation. Fortunately, cells also possess the capacity to manage some level of miscoding and SCR by degrading readthrough products (Arribere et al, 2016).…”

Transcriptomic and Proteostasis Networks of CFTR and the Development of Small Molecule Modulators for the Treatment of Cystic Fibrosis Lung Disease

“…Currently, synthetic aminoglycosides, ataluren derivatives, and escin, the FDA-approved active component of horse chestnut seed, are being investigated as readthrough agents in W1282X/Phe508del-CFBE and human primary epithelial cells [182][183][184][185]. It is important to note that such agents may cause the insertion of non-native amino acids at the site of readthrough, which may reduce channel function [186]. Currently, ELX-02, a eukaryotic ribosomal selective glycoside developed by Eloxx Pharmaceuticals (Waltham, MA, USA), is in Phase 2 clinical trials as a premature stop codon readthrough agent (NCT04135495).…”

A Review of Gene, Drug and Cell-Based Therapies for Usher Syndrome

“…However, they are associated with nephrotoxic (Mingeot-Leclercq and Tulkens, 1999) and ototoxic (Selimoglu, 2007) effects and often result in the incorporation of a non-cognate amino acid, which may alter protein function. A recent advancement in the field was the development of anticodon engineered transfer RNAs (ACE-tRNAs), which recognize and suppress stop codons while encoding the cognate amino acid of the non-mutant polypeptide (Lueck et al, 2019). This method induced a specific readthrough of CFTR mutations in vitro.…”