Exogenous control of mammalian gene expression via modulation of translational termination George J Murphy, Gustavo Mostoslavsky, Darrell N Kotton & Richard C Mulligan Here, we describe a system for the exogenous control of gene expression in mammalian cells that relies on the control of translational termination. To achieve gene regulation, we modified protein-coding sequences by introduction of a translational termination codon just downstream from the initiator AUG codon. Translation of the resulting mRNA leads to potent reduction in expression of the desired gene product. Expression of the gene product can be controlled by treating cells that express the mRNA with either aminoglycoside antibiotics or several nonantibiotic compounds. We show that the extent of regulation of gene expression can be substantial (60-fold) and that regulation can be achieved in the case of a variety of different genes, in different cultured cell lines and in primary cells in vivo. This gene regulation strategy offers significant advantages over existing methods for controlling gene expression and should have both immediate experimental application and possible clinical application. The ability to exogenously control the expression of genes in mam- malian cells has been a powerful tool of biomedical research 1–4 . In particular, gene regulation technology has played a key part in efforts to understand the role of specific gene products in fundamental biological processes and in both normal development and disease states 1 . It is likely that this form of genetic technology will continue to have an impact on a variety of areas of basic research and may even enable new therapeutic paradigms, such as the regulated delivery of protein therapeutics 3 . The technology may also have significant future impact upon the safety of gene therapy strategies. To date, most of the gene regulation systems commonly used are based on the control of transcription 1 . In spite of their considerable utility, these systems possess some significant limitations because of their reliance on chimeric transcriptional transactivators and specia- lized promoter elements. Such limitations include the requirement for co-introduction of genes encoding the relevant transcriptional trans- activator along with the gene to be regulated, and the inability to provide for the ‘on-off regulation’ of a gene in the context of its own endogenous transcriptional control elements. More recently, several RNA-only strategies for the control of gene expression have been developed that may overcome some of the limitations of transcription-based gene regulation systems. Such strategies rely on either RNA interference or siRNA technology 5 or ribozyme-mediated RNA self-cleavage 6 . Here, we provide proof of principle for another RNA-only gene regulation strategy that relies on the control of translational termination. We show that the extent of induction of expression of gene products obtainable using this strategy can be substantial, that the induction of expression is rapid and that regulation of expression can be achieved in both in vitro and in vivo contexts. This system should extend the experimental utility of existing systems for the regulation of genes in mammalian cells and may have important clinical application in the setting of gene therapy protocols. RESULTS Design features of translation-based gene regulation system The strategy for controlling gene expression via the modulation of translational termination is shown in Figure 1a. First, a translational termination (nonsense) codon is introduced into transgene coding sequences, close to the AUG codon that serves to initiate translation of the complete protein. Upon introduction of vectors encoding trans- gene sequences into cells, translation of the resulting mRNA should result in production of only a short nonfunctional peptide. Addition of small molecules capable of suppressing translational termination should result in production of the desired full-length protein. Based on previous studies that have shown that aminoglycoside antibiotics are capable of suppressing nonsense mutations in mammalian cell lines and in animal models 7–11 , and may act similarly in humans 12 , we expected that aminoglycoside antibiotics could serve as inducers of gene expression in such a system. We chose G418 (or Geneticin) 13 for our initial studies, based on a number of published reports that it was the most effective aminoglycoside for suppressing nonsense mutations in mammalian cells. 7,10,11 To prevent the constitutive generation of truncated transgene products that might engender immune responses in vivo, we positioned nonsense codon sequences very close to the initiator AUG, such that translation termination would result in the production of a short two-to-three amino-acid peptide, a peptide size insufficient for classic antigen presentation 14 . Such a configuration of sequences was modeled after a naturally occurring nonsense mutation in the human gene encoding apolipoprotein CII (APOC2 Paris2 ), which results in the near-complete loss of the corresponding gene product in an Received 3 November 2005; accepted 10 February 2006; published online 6 August 2006; doi:10.1038/nm1376 Department of Genetics, Harvard Medical School, and Division of Molecular Medicine, Children’s Hospital, Boston, Massachusetts 02115, USA. Correspondence should be addressed to R.C.M. (mulligan@receptor.med.harvard.edu). NATURE MEDICINE VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2006 1093 TECHNICAL REPORTS © 2006 Nature Publishing Group http://www.nature.com/naturemedicine