Gene selective suppression of nonsense termination using antisense agents

Biochim Biophys Acta. 2005 Sep 25;1730(3):165-72. doi: 10.1016/j.bbaexp.2005.07.004.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Codon, Nonsense* / chemistry
  • Codon, Nonsense* / drug effects
  • Codon, Nonsense* / genetics
  • Dose-Response Relationship, Drug
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Genes, Bacterial* / drug effects
  • Genes, Bacterial* / genetics
  • Genes, Reporter
  • Genes, Suppressor* / drug effects
  • Gentamicins / pharmacology
  • Luciferases / analysis
  • Luciferases / genetics
  • Mutation* / drug effects
  • Mutation* / genetics
  • Paromomycin / pharmacology
  • Plasmids
  • RNA, Messenger / analysis
  • RNA, Messenger / drug effects
  • RNA, Messenger / metabolism

Substances

  • Anti-Bacterial Agents
  • Codon, Nonsense
  • Gentamicins
  • RNA, Messenger
  • Paromomycin
  • Luciferases