Gene selective suppression of nonsense termination using antisense agents.
An estimated one third of all inherited genetic disorders and many forms of cancer are caused by premature (nonsense) termination codons. Aminoglycoside antibiotics are candidate drugs for a large number of such genetic diseases; however, aminoglycosides are toxic, lack specificity and show low efficacy in this application. Because translational termination is an active process, we considered that steric hindrance by antisense sequences could trigger the ribosome's "default mode" of readthrough when positioned near nonsense codons. To test this hypothesis, we performed experiments using plasmids containing a luciferase reporter with amber, ochre and opal nonsense mutations within the luxB gene in Escherichia coli. The nonspecific termination inhibitors gentamicin and paromomycin and six antisense peptide nucleic acids (PNA) spanning the termination region were tested for their potential to suppress the luxB mutation. Gentamicin and paromomycin increased luciferase activity up to 2.5- and 10-fold, respectively. Two of the PNAs increased Lux activity up to 2.5-fold over control levels, with no significant effect on cell growth or mRNA levels. Thus, it is possible to significantly suppress nonsense mutations within target genes using antisense PNAs. The mechanism of suppression likely involves enhanced readthrough, but this requires further investigation. Nonsense termination in human cells may also be susceptible to suppression by antisense agents, providing a new approach to address numerous diseases caused by nonsense mutations.
Kulyté
Agné
A
Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, 171 77 Stockholm, Sweden. agne.kulyte@sh.se
Dryselius
Rikard
R
Karlsson
Jenny
J
Good
Liam
L
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018389:Codon, Nonsense / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter; D016153:Genes, Suppressor / Q000187:drug effects; D005839:Gentamicins / Q000494:pharmacology; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D010303:Paromomycin / Q000494:pharmacology; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects / Q000378:metabolism
D000900:Anti-Bacterial Agents; D018389:Codon, Nonsense; D005839:Gentamicins; D012333:RNA, Messenger; D010303:Paromomycin; D008156:Luciferases