Exogenous control of mammalian gene expression through modulation of RNA self-cleavage

Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

[1]  A. Aszalos,et al.  Identification of antibiotic 1037 as toyocamycin. , 1966, The Journal of antibiotics.

[2]  G D Stormo,et al.  Sequence requirements of the hammerhead RNA self-cleavage reaction. , 1990, Biochemistry.

[3]  T. Cech.  Self-Splicing and Enzymatic Activity of an Intervening Sequence RNA from Tetrahymena , 1990, Bioscience reports.

[4]  Self‐Splicing and Enzymatic Activity of an Intervening Sequence RNA from Tetrahymena , 1990 .

[5]  T. Braun,et al.  The muscle regulatory gene, Myf-6, has a biphasic pattern of expression during early mouse development , 1991, The Journal of cell biology.

[6]  J. Davies,et al.  Antibiotic inhibition of group I ribozyme function , 1991, Nature.

[7]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R Cedergren,et al.  Numbering system for the hammerhead. , 1992, Nucleic acids research.

[9]  M. Rudnicki,et al.  Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death , 1992, Cell.

[10]  I. Nonaka,et al.  Myogenin gene disruption results in perinatal lethality because of severe muscle defect , 1993, Nature.

[11]  M. Rudnicki,et al.  MyoD or Myf-5 is required for the formation of skeletal muscle , 1993, Cell.

[12]  Y Wang,et al.  A regulatory system for use in gene transfer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Mcswiggen,et al.  In vitro and in vivo comparison of hammerhead, hairpin, and hepatitis delta virus self-processing ribozyme cassettes. , 1994, The Journal of biological chemistry.

[14]  S. Tajbakhsh,et al.  Lineage restriction of the myogenic conversion factor myf-5 in the brain. , 1995, Development.

[15]  O. Uhlenbeck,et al.  Inhibition of the hammerhead ribozyme by neomycin. , 1969, RNA.

[16]  T. Ley,et al.  Long-range disruption of gene expression by a selectable marker cassette. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[17]  B. Wold,et al.  Know Your Neighbors: Three Phenotypes in Null Mutants of the Myogenic bHLH Gene MRF4 , 1996, Cell.

[18]  Shannon R. Magari,et al.  A humanized system for pharmacologic control of gene expression , 1996, Nature Medicine.

[19]  R. Mulligan,et al.  A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  S. Tajbakhsh,et al.  Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice , 1996, Nature.

[21]  Antibiotic interactions with the hammerhead ribozyme:tetracyclines as a new class of hammerhead inhibitor. , 1996, The Biochemical journal.

[22]  R. Jaenisch,et al.  Myogenin can substitute for Myf5 in promoting myogenesis but less efficiently. , 1997, Development.

[23]  G. Cossu,et al.  Redefining the Genetic Hierarchies Controlling Skeletal Myogenesis: Pax-3 and Myf-5 Act Upstream of MyoD , 1997, Cell.

[24]  M. Zillmann,et al.  In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions. , 1997, RNA.

[25]  M. Rudnicki,et al.  MyoD and Myf-5 differentially regulate the development of limb versus trunk skeletal muscle. , 1997, Development.

[26]  S. Koester,et al.  Ectopic Pax-3 Activates MyoD and Myf-5 Expression in Embryonic Mesoderm and Neural Tissue , 1997, Cell.

[27]  E. Westhof,et al.  Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme. , 1998, Chemistry & biology.

[28]  David K. Stevenson,et al.  Bioluminescent indicators in living mammals , 1998, Nature Medicine.

[29]  F. Gage,et al.  High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Wei Zhang,et al.  Overlapping functions of the myogenic bHLH genes MRF4 and MyoD revealed in double mutant mice. , 1998, Development.

[31]  E. Westhof,et al.  Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA. , 1998, Journal of molecular biology.

[32]  P. Baldacci,et al.  Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation , 1998, Mammalian Genome.

[33]  Robert Cedergren,et al.  Schistosome Satellite DNA Encodes Active Hammerhead Ribozymes , 1998, Molecular and Cellular Biology.

[34]  J. Szostak,et al.  In vitro selection of functional nucleic acids. , 1999, Annual review of biochemistry.

[35]  M. Rudnicki,et al.  Myogenic determination occurs independently in somites and limb buds. , 1999, Developmental biology.

[36]  T. Lockett,et al.  Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration. , 1999, Nucleic acids research.

[37]  J. Moran,et al.  Limbs move beyond the Radical fringe , 1999, Nature.

[38]  R Cedergren,et al.  Hammerhead-mediated processing of satellite pDo500 family transcripts from Dolichopoda cave crickets. , 2000, Nucleic acids research.

[39]  Philippe Soriano,et al.  Early myotome specification regulates PDGFA expression and axial skeleton development. , 2000, Development.

[40]  S. Tajbakhsh,et al.  The birth of muscle progenitor cells in the mouse: spatiotemporal considerations. , 2000, Current topics in developmental biology.

[41]  T. Braun,et al.  Myf-5 Revisited Loss of Early Myotome Formation Does Not Lead to a Rib Phenotype in Homozygous Myf-5 Mutant Mice , 2000, Cell.

[42]  S. Tapscott,et al.  Molecular Distinction between Specification and Differentiation in the Myogenic Basic Helix-Loop-Helix Transcription Factor Family , 2001, Molecular and Cellular Biology.

[43]  Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology , 2001, Nature Biotechnology.

[44]  P. Morcos.  Achieving efficient delivery of morpholino oligos in cultured cells , 2001, Genesis.

[45]  P. Rigby,et al.  A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development. , 2001, Development.

[46]  Ronald R. Breaker,et al.  Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression , 2002, Nature.

[47]  R. Breaker.  Engineered allosteric ribozymes as biosensor components. , 2002, Current opinion in biotechnology.

[48]  P. Rigby,et al.  Expression of the myogenic regulatory factor Mrf4 precedes or is contemporaneous with that of Myf5 in the somitic bud , 2002, Mechanisms of Development.

[49]  David R Corey,et al.  Novel antisense and peptide nucleic acid strategies for controlling gene expression. , 2002, Biochemistry.

[50]  M. Rudnicki,et al.  Myf5 and MyoD activation define independent myogenic compartments during embryonic development. , 2003, Developmental biology.

[51]  S. Tajbakhsh.  Stem cells to tissue: molecular, cellular and anatomical heterogeneity in skeletal muscle. , 2003, Current opinion in genetics & development.

[52]  S. Silverman,et al.  Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA. , 2003, RNA.

[53]  R. Breaker,et al.  Adenine riboswitches and gene activation by disruption of a transcription terminator , 2004, Nature Structural &Molecular Biology.

[54]  R. Breaker,et al.  Control of gene expression by a natural metabolite-responsive ribozyme , 2004, Nature.

[55]  T. Cech.  RNA finds a simpler way , 2004, Nature.

[56]  V. Luria,et al.  Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase , 1998, Transgenic Research.