Biology subjects

Porter, J. J.

Publications and source records attributed to Porter, J. J..

6 recordsLinked to original sources

bioRxiv2025

Anticodon Edited Transfer RNAs (ACE-tRNAs) Encoded as Therapeutic Nonviral Minimal DNA Vectors

Nonsense mutations, resulting from a premature termination codon (PTC), make up [~]11% of all genetic lesions causing disease, affecting millions of people worldwide. Nonsense suppressor anticodon-edited tRNAs (ACE-tRNAs) have emerged as a therapeutic modality for the rescue of PTCs. Delivery of ACE-tRNAs in vivo has been achieved by adeno-associated viral vector and RNA-lipid nanoparticle, however due to drawbacks associated with these approaches, DNA delivery remains an attractive approach. DNA-based approaches afford ease of manufacturing at a relatively low cost and exhibit improved therapeutic durability and safety as compared to viral vector-or RNA-based approaches. Due to the small size of human tRNA genes employed as ACE-tRNAs, in principle, DNA vectors <200 base pairs (bp) in size (minivectors) could be utilized for delivery of actively transcribed ACE-tRNAs. Here we demonstrate that linear DNA ACE-tRNA vectors as small as 200 bp effectively suppress several nonsense mutations in CFTR and REP1, and that ACE-tRNA minivectors display significantly improved bioavailability, reduced innate immune burden, and superior biostability as compared to conventional plasmid DNA vectors.

molecular biology↗

bioRxiv2025

Anticodon-edited tRNA enables translational readthrough of COL4A5 premature termination codons

Alport syndrome is caused by variants in COL4A3, COL4A4, or COL4A5, which encode the 345 chains of type IV collagen. These variants result in defects in the glomerular basement membrane (GBM) and impaired kidney function. Nonsense variants result in truncated proteins lacking the NC1 domain, thereby preventing proper GBM assembly and function and causing the most severe forms of the disease. Restoring full-length protein expression represents a potential therapeutic strategy for Alport syndrome and related disorders. Anticodon-edited transfer RNAs (ACE-tRNAs), which promote premature termination codon (PTC) readthrough, have shown promise in diseases such as cystic fibrosis, but their application in Alport syndrome remains unexplored. To assess the potential of ACE-tRNAs for PTC readthrough of COL4A5 nonsense variants, we employed a C-terminal NanoLuc-fused COL4A5 reporter system in which luminescence is produced only upon full-length protein translation. We introduced ACE-tRNAs into HeLa and 293T cells expressing one of four COL4A5 nonsense variants (S36X, R1563X, S1632X, and R1683X) identified in patients with X-linked Alport syndrome. Readthrough efficiency was evaluated via NanoLuc luminescence and western blotting. Furthermore, we assessed the efficiency of ACE-tRNA-restored 345 heterotrimer formation using a split NanoLuc-based assay. Our results show that application of ACE-tRNAs led to restored C-terminal luminescence across all four COL4A5 nonsense variants, indicating successful readthrough and full-length translation. Moreover, the restored COL4A5 proteins formed 345 heterotrimers. These findings support ACE-tRNA-mediated nonsense suppression as a promising therapeutic strategy for Alport syndrome, with the potential to restore GBM integrity in patients harboring nonsense variants.

genetics↗

bioRxiv2024

Gz Enhanced Signal Transduction assaY (GZESTY) for GPCR deorphanization

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

pharmacology and toxicology↗

bioRxiv2024

Anticodon Engineered Transfer RNAs (ACE-tRNAs) are a Platform Technology for Suppressing Nonsense Mutations

Nonsense mutations arise from single nucleotide substitutions that result in premature termination codons (PTCs). PTCs result in little to no full-length protein production and loss of mRNA expression through the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate that anticodon engineered (ACE-) tRNAs efficiently suppress the most prevalent cystic fibrosis (CF) causing PTCs, promoting significant rescue of endogenous cystic fibrosis transmembrane conductance regulator (CFTR) transcript abundance and channel function in different model systems. We demonstrate that our best-performing ACE-tRNA, that decodes all UGA PTCs to a leucine amino acid, markedly rescues CFTR channel function from the most prevalent CF causing PTCs that arise from non-leucine encoding codons. Using this single ACE-tRNA variant, we demonstrate significant rescue of CFTR channel function in an immortalized airway cell line and two different primary CF patient-derived intestinal cell models with CFTR nonsense mutations. Thus, ACE-tRNAs have promise as a platform therapeutic for CF and other nonsense-associated diseases.

molecular biology↗

bioRxiv2024

Optimization of Anticodon Edited Transfer RNAs (ACE-tRNAs) Function in Translation for Suppression of Nonsense Mutations

Nonsense suppressor tRNAs or AntiCodon-Edited tRNAs (ACE-tRNAs) have long been envisioned as a therapeutic approach to overcome genetic diseases resulting from the introduction of premature stop codons (PTCs). The ACE-tRNA approach for the rescue of PTCs has been hampered by ineffective delivery through available modalities for gene therapy. Here we have screened a series of ACE-tRNA expression cassette sequence libraries containing >1,800 members to optimize ACE-tRNA function and provide a roadmap for optimization in the future. By optimizing PTC suppression efficiency of ACE-tRNAs, we have decreased the amount of ACE-tRNA required by [~]16-fold for the most common cystic fibrosis-causing PTCs.

molecular biology↗

bioRxiv2021

Efficient Suppression of Endogenous CFTR Nonsense Mutations Using Anticodon Engineered Transfer RNAs

Nonsense mutations or premature termination codons (PTCs) comprise [~]11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases, however their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542X-, R1162X- and W1282X-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential stand-alone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC containing endogenous genes. One Sentence SummarySuppression of endogenous CFTR nonsense mutations by anticodon engineered tRNAs significantly increases mRNA expression and channel function.

molecular biology↗