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A nonsense mutation results in the expression of a truncated protein, and therapeutic strategies aim to restore full-length protein expression. Most strategies under development, including small-molecule aminoglycosides, suppressor tRNAs, or the targeted degradation of termination factors, lack mRNA target selectivity and may poorly differentiate between nonsense and normal stop codons, resulting in off-target translation errors. Here, we demonstrate that antisense oligonucleotides can stimulate readthrough of disease-causing nonsense codons, resulting in high yields of full-length protein in mammalian cellular lysate. Readthrough efficiency depends on the sequence context near the stop codon and on the precise targeting position of an oligonucleotide, whose interaction with mRNA inhibits peptide release to promote readthrough. Readthrough-inducing antisense oligonucleotides (R-ASOs) enhance the potency of non-specific readthrough agents, including aminoglycoside G418 and suppressor tRNA, enabling a path toward target-specific readthrough of nonsense mutations in CFTR, JAG1, DMD, BRCA1 and other mutant genes. Finally, through systematic chemical engineering, we identify heavily modified fully functional R-ASO variants, enabling future therapeutic development.<\/jats:p>","DOI":"10.1093\/nar\/gkae624","type":"journal-article","created":{"date-parts":[[2024,7,16]],"date-time":"2024-07-16T10:51:59Z","timestamp":1721127119000},"page":"8687-8701","source":"Crossref","is-referenced-by-count":12,"title":["mRNA-specific readthrough of nonsense codons by antisense oligonucleotides (R-ASOs)"],"prefix":"10.1093","volume":"52","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-5925-9459","authenticated-orcid":false,"given":"Denis","family":"Susorov","sequence":"first","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , Worcester , MA 01605 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1040-0855","authenticated-orcid":false,"given":"Dimas","family":"Echeverria","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , Worcester , MA 01605 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6928-8071","authenticated-orcid":false,"given":"Anastasia","family":"Khvorova","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , Worcester , MA 01605 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1588-717X","authenticated-orcid":false,"given":"Andrei\u00a0A","family":"Korostelev","sequence":"additional","affiliation":[{"name":"RNA Therapeutics Institute, UMass Chan Medical School , 368 Plantation Street , Worcester , MA 01605 , USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,7,16]]},"reference":[{"key":"2024082703491681700_B1","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1002\/humu.20763","article-title":"A meta-analysis of nonsense mutations causing human genetic disease","volume":"29","author":"Mort","year":"2008","journal-title":"Hum. 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