@article{26e41fb0f0bc439db70251ed7539f9f2,
title = "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).",
author = "Lueck, {John D.} and Yoon, {Jae Seok} and Alfredo Perales-Puchalt and Mackey, {Adam L.} and Infield, {Daniel T.} and Behlke, {Mark A.} and Pope, {Marshall R.} and Weiner, {David B.} and Skach, {William R.} and McCray, {Paul B.} and Ahern, {Christopher A.}",
note = "Funding Information: This work and JDL were supported by Emily{\textquoteright}s Entourage (www.emilysentourage.org). Additional support was provided by a Cystic Fibrosis Foundation (CFF) Pilot Award (R458-CR11) and Research Grant (498721), the NIH (GM106568) and CAA is an American Heart Association Established investigator (5EIA22180002). J.S.-Y. and W.R.S. are supported directly through the CFF and Cystic Fibrosis Foundation Therapeutics (CFFT) Research laboratory. DTI is supported by the Cystic Fibrosis Foundation (INFIEL17F0). D.B.W. is funded by the WW Smith Family Trust. PBM and CAA are supported by the Roy J. Carver Charitable Trust. The authors acknowledge formative discussions with J. Kevin Foskett (University of Pennsylvania) that facilitated development of this study. We thank Dr. Shikha Mishra at ThermoFisher for fruitful nucleotide delivery discussion and providing reagents used in this study. We thank Dr. Julien Sebag (University of Iowa, Department of Molecular Physiology and Biophysics) for providing access to Spectramax i3. We thank Grace Galles and Selena Borrill for their technical assistance. We acknowledge Inovio Pharmaceuticals for the use of the CELLECTRA 3P for in vivo electroporation. Approved by Institutional Animal Care and Use Committee at the Wistar Institute (protocol number: 112762). Funding Information: Competing interests: D.B.W. receives research funding from Inovio Pharmaceuticals, and from GeneOne Pharmaceuticals. He has received Honaria for speaking at Merck, Roche & AstraZeneca, has ownership interest (including patents) in Inovio Pharmaceuticals and has been a consultant/advisory board member for Inovio Pharmacueticals and Gene One ((PCT/US2018/059065, filed November 2, 2018; METHODS OF RESCUING STOP CODONS VIA GENETIC REASSIGNMENT WITH ACE-tRNA; Inventors - University of Iowa - C.AA. and J.D.L.; Pertains to the tRNA sequences in Figure 2, Supplementary Figure 2a and Supplementary Data 1 and Data 2); (PCT/US2018/59085, filed November 2, 2018; METHODS OF RESCUING STOP CODONS VIA GENETIC REASSIGNMENT WITH ACE-tRNA; Inventors - The Wistar Institute of Anatomy and Biology, University of Iowa - A.P.-P., J.D.L., D.B.W. and C.A.A.; Pertains to in vivo delivery data shown in Figure 5. The remaining authors declare no competing interests.",
year = "2019",
month = dec,
day = "1",
doi = "10.1038/s41467-019-08329-4",
language = "English (US)",
volume = "10",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}