Data demonstrate that U6 promoter-driven and single-stranded AAV2/9 constructs show variable and dose-dependent activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100-bp genomic context provides broad and efficacious PTC rescue.
Abstract
Abstract Protein-truncating variants caused by stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases, with UGA codons being most common. Suppressor transfer RNA (sup-tRNA) has therapeutic potential for premature termination codon (PTC) rescue but has thus far underperformed by traditional AAV delivery platforms, and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to optimize viral payloads with sup-tRNA genes. These data demonstrate that U6 promoter-driven and single-stranded AAV2/9 constructs show variable and dose-dependent activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100-bp genomic context provides broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV-delivered ArgUCA sup-tRNA in brain demonstrates no effects on endogenous tRNA levels, their acylation, or processing, and these features are also maintained in the delivered ArgUCA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA PTC stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain.
Nonsense variants cause 18% of inherited retinal diseases (IRDs), yet current therapies require variant-specific development, leaving most patients untreated. Here, we combined a large-scale genetic analysis literature survey of >37,500 IRD patients with anticodon-edited (ACE)-tRNA engineering to create a single, gene-agnostic therapy targeting Arg>Ter nonsense variants which are the most prevalent subclass (35%) of premature stop codons (PTCs). We developed an optimized ACE-tRNA (V3) that achieved up to 86% readthrough across 13 clinically relevant variants, restored native PRCD localization in the arRP-causing p.R22* mutant, and demonstrated activity in photoreceptor-like cells. To enable translation, we established an AAV2/7m8 production platform (1*10¹²–1*10¹³ GC/mL) and defined 1*10 GC/eye as the safe dose in mice. This patient genetics-guided strategy positions ACE-tRNA_V3 as a promising candidate for preclinical development, offering a precision medicine approach that targets the most common nonsense variant class with a single therapeutic molecule.
Asodu Sandeep Sarma, Alaa Saleh, Jonathan Eintracht et al.· bioRxiv· 0 citations
Suppressor transfer RNAs (sup-tRNAs) can rescue disease-causing nonsense mutations by promoting readthrough of premature termination codons (PTCs). Their clinical translation is limited by suboptimal activity and inefficient in vivo delivery. In this work, we combined site-specific chemical modification of sup-tRNAs with cargo-tailored pulmonary lipid nanoparticle (LNP) engineering to overcome these barriers. Incorporation of N1-methyladenosine in sup-tRNAs improved PTC readthrough, enhanced tRNA aminoacylation, prolonged functional persistence, and reduced innate immune activation. High-throughput ionizable lipid screening and formulation optimization identified a sup-tRNA-tailored LNP that efficiently delivered chemically modified sup-tRNAs to the lung. This approach restored cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in bronchial epithelial cells, mouse models, and patient-derived organoids. Thus, LNP-delivered, chemically engineered sup-tRNAs represent a potential therapeutic platform for treating nonsense mutations.
Programmed translational readthrough produces C-terminally extended protein isoforms via decoding of stop codons by near-cognate tRNAs. Human genes experimentally validated as readthrough targets share a CUAG motif downstream of a UGA stop codon. However, the full sequence determinants of readthrough efficiency, how they combine, and how generalisable they are across genes remain largely unexplored. Here we use deep mutational scanning to quantify ∼1,400 sequence variants for each of the three examples of human readthrough in the genes AQP4, MAPK10 and OPRK1. In addition to the core CUAG motif, mutations that modulate readthrough elements extend up to +27 nucleotides downstream of the stop codon and across six codons (18 nucleotides) upstream. For the downstream sequence, an additive model with a sigmoidal global epistasis function captures most of the within-gene readthrough variance for double mutants (R²=0.84-0.96), with additional contributions from a small number of strong pairwise interactions. Mutational effects nonetheless generalise poorly between genes: only the immediate -3 to +4 nucleotide window shows consistent behaviour, while mutations in more distal positions have context-dependent effects. Combinatorial assembly of sequence blocks from different genes into chimeras reveals strong interactions (epistasis) between sequences upstream and downstream of the stop codon. This study provides comprehensive quantitative maps of the sequence determinants of human programmed readthrough and suggests that three examples of programmed readthrough are located on distinct local fitness peaks, each defined by different upstream and downstream architectures built around a shared CUAG core motif.
Ignasi Toledano, Fran Supek, Ben Lehner· bioRxiv· 0 citations
Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) removes ~100 amino acids and accounts for approximately 10% of RTT-causing mutations. Their pathogenicity is unexpected because this C-terminal domain is dispensable in mice. Analysis of pathogenic and benign human MECP2 variants reveals that some individuals with apparently typical CTDs do not develop Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human sequence data and mouse models we show that pathogenicity results from a marked reduction in MeCP2 levels and depends on the presence of a proline proline stop motif (-PPX) generated by a shift to the +2 reading frame. CTDs that shift to the +1 frame avoid this motif and are benign. Replacing the stop codon of the PPX motif with tryptophan restores MeCP2 expression and rescues RTT-like phenotypes in a CTD mouse model. An adenine base editor efficiently introduces this substitution in cultured cells. These findings define a reliable prognostic distinction between benign and pathogenic CTDs and establish a potential editing strategy for correcting disease-causing CTD mutations.
Jacky Guy, E. Hein, Beatrice K. Alexander-Howden et al.· eLife· 0 citations
The CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases as well as the preferential advantages of CRISPR over the antisense technology recently developed targeting the PE of EZH2.
Overall, the findings suggest that TA is not a universal response to loss-of-function mutations in yeast, and the ability to resist specific transcriptomic ruptures would thus rely mostly on the general robustness of genetic networks.
Marzena Marszałek, W. Babik, R. Korona et al.· Molecular biology and evolut...· 0 citations