EP1224305A2 - Selbstschneidende rna-sequenzen und deren verwendung in der kontrole der proteinsynthese - Google Patents
Selbstschneidende rna-sequenzen und deren verwendung in der kontrole der proteinsyntheseInfo
- Publication number
- EP1224305A2 EP1224305A2 EP00977426A EP00977426A EP1224305A2 EP 1224305 A2 EP1224305 A2 EP 1224305A2 EP 00977426 A EP00977426 A EP 00977426A EP 00977426 A EP00977426 A EP 00977426A EP 1224305 A2 EP1224305 A2 EP 1224305A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- ligand
- gene
- rna
- self
- cleavage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1048—SELEX
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
Definitions
- This invention relates to the control of protein synthesis from a mRNA sequence (activation or repression) by inserting a sequence into an untranslated region (UTR) of a gene which confers conditional self-cleavage to the transcribed RNA.
- UTR untranslated region
- the first level is the transcription of the gene to generate a pre-messengerRNA (pre-mRNA).
- pre-mRNA pre-messengerRNA
- the rate of transcription initiation as well as the transcript elongation are under tight physiological control by the basal transcription machinery.
- Another level is pre-mRNA splicing into a mature mRNA.
- Alternative splicing events can occur in tissue-specific manner or under hormonal control, hence the active protein is synthesized in a restricted set of tissue or physiological environment.
- Other levels of control are the mRNA turn-over and the ability of the mRNA to be translated into protein. Hence drug-control of protein synthesis can theoretically be addressed at each of the aforementioned levels.
- a drug can control the expression of a target gene within a genome by altering the rate of synthesis of the RNA. It can be achieved by drugs which bind DNA with high affinity and sequence-specificity and hence compete with the transcription factors necessary for the transcription of the target gene.
- drugs may be triple helix-forming oligonucleotides (C. Helene et al., Ciba Found Symp. (1997) 209: 94-106), peptide nucleic acids (Pooga, M. et al.; Nat Biotechnol. (1998) 16(9):857-61) or pyrrole- imidazole polyamides (Dickinson, L.A. et al.; Proc Natl Acad Sci U S A.
- Ribozymes anneal with a target RNA and cleave it (Forster, A.C. & Altaian, S.; Science (1990) 249: 783-6).
- Ribozymes are typically RNA molecules which have enzyme-like catalytic activities usually associated with cleavage, splicing or ligation of nucleic acid sequence.
- the typical substrates for catalytically active ribozymes are RNA molecules, although ribozymes can catalyze reactions in which DNA molecules serve as substrates.
- ribozymes which are active intracellularly work in cis, catalyzing only a single turnover reaction, and are usually self-modified during the reaction (Cech, T.R.; Biosci Rep. 1990; 10(3):239-61 ).
- ribozymes can be engineered to act in trans, in a truly catalytic manner, with a turnover greater than one and without being self-modified.
- Two distinct regions can be identified in a ribozyme: the binding region which gives the ribozyme its specificity through hybridization to a specific nucleic acid sequence, and a catalytic region which gives the ribozyme the activity of cleavage, ligation or splicing.
- Drug-control of the protein production from a specific target gene within a genome can be addressed by controlling the translation of the mRNA into protein.
- Control of gene expression which takes advantage of the interaction of a specific ligand- binding RNA (aptamer) and its cognate ligand has been published (Werstuck, G. & Green, M.R.; Science (1998) 282, 296-298).
- Translation of the mRNA is inhibited by a drug which binds in the vicinity of the 5' cap structure of the mRNA. In this setting, the intracellular presence of the drug is repressing protein production.
- the control of gene expression and protein production is advantageous in the field of gene therapy and DNA vaccines. It is intended to prevent the occurrence of adverse side effects and to tune the level of expression within an efficient therapeutic window.
- DNA binding drugs as well as anti- sense and ribozyme oligonucleotides have a poor bio-availability and are readily degraded by nucleases and proteases within tissues or body fluids.
- Xenogenic transcription factors may elicit a cytotoxic immune response which will destroy the cells expressing the transgenes and hence undermine the therapy or the vaccination (S. K. Tripathy et al., Nature Medicine (1996) 2, 545-550).
- the drugs which activate the xenogenic transcription factors are antibiotics, estrogen analogs or immuno- suppressors. They are not therapeutics per se for the gene therapy and may have undesired side effects.
- an inducer drug which is at least innocuous, for the therapy considered.
- An additional drawback is the large cloning space needed in the vectors, in order to accommodate the control sequences and the genes encoding the transcription factors. Therefore it is highly advantageous i) to avoid the use of xenogenic proteins for the control of gene expression and ii) to choose the inducer drug according to the therapeutic objectives.
- Control of gene expression and protein production is advantageous in the field of functional genomics, transgenic plants and transgenic animals.
- the conditional expression of a gene into cell cultures or whole plants or animals and the comparison of the phenotypes with and without gene expression enables one to decipher the function of the gene.
- the drawbacks of the xenogenic transcription factor-based gene switches are two-fold: i) the expression of the target gene and the translation of the mRNA are no more under control of the endogenous physiological stimuli and ii) the effect of the inducer drug on the physiology of the cells. Therefore it is highly advantageous i) to keep the transgene under the control of its endogenous transcription promoter, as well as maintain the same translation control sequences on the mRNA and ii) to choose an inducer drug which does not alter cell physiology.
- genes according to the instant invention can be any nucleic acid encoding a biological product, comprising one or more naturally present or artifically produced untranslated region(s).
- Untranslated region (UTR) of a gene :
- the UTR may be located before the 5' -end of the coding region (5'-UTR), after the 3'-end of the coding (3'-UTR), or may be an intron inserted within the coding region.
- the UTR may be located before the 5 '-end of the coding region (5'-UTR), after the 3 '-end of the coding (3'-UTR), or may be an intron inserted within the coding region.
- the ligand can be a nucleic acid molecule, a protein, polysaccharide or sugar, or an organic or inorganic molecule which interacts with the self-cleaving RNA sequence and either inhibits or stimulates self-cleavage.
- the instant invention provides methods and compositions for the control of protein production in a cell. More particularly, the invention provides methods and compositions for the control of protein production using particular nucleic acid sequences, inserted within at least one UTR of a gene, which confer ligand-dependant self-cleavage to RNAs transcribed from said gene. Hence, the pre-mRNA or the mRNA can undergo self-cleavage that can be regulated through the presence, or the absence of a ligand. The ligand-controlled cleavage enables one to control protein production with this ligand (figure 1). This method is useful in situations where a gene is transfected into cultured cells or into live animals and one needs to control the expression of this gene.
- the amount of a protein synthesized in a cell is proportional to the amount of its mRNA, and depends on efficient translation of its mRNA into protein.
- Two features of mRNAs are essential for their efficient translation into proteins (Gallie, R.; Genes & Dev. (1991) 5: 2108-2116) and for their stability (Beelman, CA. & Parker, R.; Cell (1995) 81: 179-183): the 5 '-cap structure and the 3'-polyA tail.
- Figure 2A shows that the expression of the luciferase gene can be controlled by removing either 3'-polyA or 5 '-cap from the mRNA.
- an active nucleic acid sequence of this invention in the 5'- or 3'-UTR of the reporter gene entails the cleavage of the transcribed RNA and hence the removal of, respectively, the 5 '-cap and 3'-polyA.
- the active nucleic acid sequence; in either location; decreases the luciferase protein production whereas an inactive nucleic acid sequence in the same location does not affect gene expression.
- This invention can be used to control production of essentially all types of proteins, in essentially all types of cells, in vitro ex vivo or in vivo.
- a first object of this invention resides more particularly in a modified gene encoding a protein, polypeptide or peptide, wherein said modified gene contains, inserted in an untranslated region thereof (UTR), a nucleic acid sequence conferring ligand- dependant self-cleavage to a RNA molecule transcribed from the gene.
- the modified gene can be a genomic DNA, a cDNA or a synthetic DNA.
- the modified gene comprises at least one UTR region, which can be naturally present in said gene or artificially inserted therein. For instance, where the gene is a gDNA molecule, the gene comprises naturally-ocurring UTRs such as 5' -UTR, introns and 3 '-UTRs.
- UTR regions may be introduced, such as introns. Furthermore, even where natural UTRs are present within the gene, additional UTR can be inserted, in addition thereto or in replacement thereof.
- the modified gene may further comprise a transcriptional promoter, to direct transcription of the coding region into pre-mRNA.
- the gene may, in addition, be contained in a vector, such as a plasmid, a virus, a cosmid, a phage, an artificial chromosome, etc.
- the modified gene may encode any type of protein, polypeptide or peptide, including protein, polypeptide or peptide of human, other mammalian, plant, viral or bacterial origin, or derivatives thereof for instance.
- the encoded biological product may exhibit therapeutic or prophylactic activity, and may also represent a marker molecule, for instance.
- the modified gene of this invention comprises at least one particular nucleic acid sequence inserted in a UTR of said gene.
- the UTR can be a 5'- UTR, a 3'-UTR or an intron.
- the modified gene may comprise several copies of such a nucleic acid sequence, inserted in various UTRs or in various locations of a UTR, or different nucleic acid sequences inserted in various UTRs or in various locations of a UTR.
- the self-cleavage of the RNA sequence inserted into a UTR of the pre-mRNA is inhibited by a ligand.
- the pre-mRNA or mRNA is cleaved in the absence of ligand and protein production is decreased ; the pre-mRNA or mRNA is not cleaved in the presence of ligand and protein production is restored.
- a particular modified gene of this invention thus comprises a nucleic acid sequence which confers ligand-inhibited self-cleavage to a RNA transcribed from the gene.
- UTR of the pre-mRNA is activated by a ligand. Hence the pre-mRNA or mRNA is cleaved in the presence of ligand and protein production is repressed; the pre-mRNA or mRNA is not cleaved in the absence of ligand and protein production is restored.
- Another particular modified gene of this invention thus comprises a nucleic acid sequence which confers ligand-activated self-cleavage to a RNA transcribed from the gene.
- the invention also relates to a method of modifying a gene, comprising inserting, within at least one untranslated region of the gene, a nucleic acid sequence conferring ligand-dependent self-cleavage to a RNA transcribed from said gene.
- the nucleic acid can be inserted in various sites within a UTR sequence.
- 5' -UTR or 3' -UTR are concerned, insertion can be performed in different sites which essentially do not affect transcription of the DNA into pre-mRNA. Such sites may be determined by analyzing the sequence of a UTR, and by using restriction sites available or artificially created. Insertion may also be accomplished by recombination, mutagenesis, etc. Where introns are concerned, insertion can take place in any region which do not affect transcription.
- RNAfold Zuker, M.; Methods Mol Biol (1994) 25:267-94
- the surrounding sequence may provide the nucleic acid sequence with alternative folding pathways which lead to inactive or ligand-independent conformations (Stage-Zimmermann, T.K. RNA (1998) 4:875-889).
- Various sites of insertion of the nucleic acid sequence within a UTR can be selected according to the above methodologies.
- the particular nucleic acid sequence used in the instant invention to provide production control can be any nucleic acid sequence encoding a ligand-dependant self- cleavable RNA.
- the size of this nucleic acid sequence can vary, depending on its nature and/or origin. Generally, the nucleic acid sequence comprises between 10 and 500 base pairs, preferably below 300 base pairs. Typical such nucleic acids comprise between 20 and 200 bp.
- the nucleic acid can be of different origin.
- the nucleic acid can be derived from naturally occuring self-cleavable RNA sequences.
- RNA sequences can be prepared artificially and used according to the instant invention.
- DNAs encoding such RNAs can be prepared by conventional techniques and used to modify a gene in accordance with the instant invention.
- any DNA sequences whose RNA transcripts have self-cleaving activity may be chosen and inhibitors of self-cleavage may be identified from a library of compounds.
- an advantageous ligand is chosen first and DNA sequences whose RNA transcripts feature ligand-dependant self-cleavage are produced in vitro.
- the invention also describes a particular method that can be used to produce artificial, non-naturally ocurring ligand-dependant self-cleavable RNA and DNA encoding them.
- Said artificial, non-naturally ocurring ligand-dependant self- cleavable RNA are called aptazymes, and represent another object of the instant invention.
- the nucleic acid sequence is an artificial DNA encoding an aptazyme.
- Said aptazyme can be obtained by an in vitro evolution and selection method, as described below.
- the ligand can be selected first, and then corresponding activated or inhibited aptazymes are produced.
- the ligand can be a nucleic acid molecule, a protein, polysaccharide or sugar, or an organic or inorganic molecule.
- the nature of the ligand can be chosen to be exogenously supplied, such as some non-toxic molecule or drug which readily enters at least the cells transfected with the transgene.
- an entirely endogenous system can be designed in which the controlling ligand is a molecule (e.g., some small metabolite or macromolecule) present within the target cell.
- the ligand can be a molecule present within the target cell population and essentially absent (or present at a lower concentration) within other cell population, thereby conferring tissue selectivity to the expression.
- the ligand may be a molecule present within the target cell population, which is directly or indirectly related to a pathology or condition to be corrected, and essentially absent from cells or tissues not affected by the pathology.
- the activity of the ligand-dependant self-cleavage nucleic acid is then dependent on its binding to the metabolite or macromolecule.
- the expression of the gene is then restricted essentially to the cells which express sufficient amounts of the metabolite or macromolecule, i.e., the diseased cell population (examples of such ligands include mutated p53 molecules, activated oncogenes, etc.).
- ligands include antibiotics (e.g., doxycycline, pefloxacine, etc.), molecules which are used in humans (such as drugs, adjuvents, substitutes, etc.) and any molecule which would be innocuous in a human subject, for instance.
- antibiotics e.g., doxycycline, pefloxacine, etc.
- molecules which are used in humans such as drugs, adjuvents, substitutes, etc.
- any molecule which would be innocuous in a human subject for instance.
- This process requires a very high degree of specificity in the molecular recognition between the aptazyme and the ligand.
- Such a specificity can in principle is achieved by a nucleic acid aptamers (Famulok, M. ; Curr. Opin. Struct. Biol. (1999); 9: 324-329).
- Aptazymes that undergo ligand-dependent self-cleavage can be prepared and isolated by an in vitro evolution and selection method.
- This method is related to the SELEX technology (US Patent 5270163, incorporated therein by reference) which is a technique that allows the simultaneous screening of highly diverse combinatorial libraries of different RNA or DNA (single stranded or double stranded DNA) molecules for a particular feature.
- SELEX SELEX technology
- These features may be i) catalytic activity of a nucleic acid, ii) the ability of a nucleic acid to specifically complex a desired target molecule with high affinity and selectivity.
- the present method differs from methods which have been previously described for the selection of allosteric ribozymes (WO94/13791; WO98/08974). More specifically, a production method of this invention (as depicted in Figure 3A and 3B), comprises:
- preparing a pool of different double-stranded deoxyribonucleic acid (DNA) molecules at least part of the sequence in the molecules of the pool is a random or partially random sequence, such that said part has a different sequence in different molecules of the pool.
- a random sequence may be prepared, for example, by utilizing a nucleic acid synthesizer,
- RNA molecules transcribing the pool of DNA molecules into a pool of ribonucleic acid (RNA) molecules under conditions where no-self cleavage should occur: in the presence of ligand for the selection of ligand-inhibited self-cleaving nucleic acid sequences, or in the absence of ligand for the selection of ligand-activated self-cleaving nucleic acid sequences,
- steps 3-5 repeating steps 3-5 over a plurality of cycles, e.g. about 1-50 cycles, to eliminate the RNA sequences which are capable of self-cleavage under non-permissive conditions, albeit with low efficiency, due to multiple, non-productive conformational states, 7. adding or removing said ligand to said pool and incubating under conditions permitting self-cleavage, and
- the method of this invention further comprises:
- This method can also be used to select aptamers, i.e. ligand-binding RNA sequences.
- the molecules in the pool are comprised of an entirely random sequence with the exception of two short flanking sequences which encompass the RNA polymerase promoter and the attachment sites of the amplification primers.
- the molecules in the pool are constructed based on a known ribozyme sequence.
- the molecule may be comprised of a constant, ribozyme-derived sequence attached to a random or semi-random sequence.
- the random sequence contains between 10 and 300 nucleotides, preferably between 20 and 200, more preferably between 20 and 180 nucleotides.
- a «random» sequence it is understood that the sequence is random only as originally used in the selection process, that the product of the selection process is not random but a set of specific sequences displaying ligand-dependent self-cleaving ability.
- the initial pool may comprise a various number of DNA molecules.
- the initial pool may comprise up to 10 20 molecules or more.
- Typical pools comprise between 10 4 and 10 16 molecules.
- the ionic composition of the selection medium will be set accordingly and the temperature will be set to 37 degrees Celsius.
- mutations can be introduced in the selected DNA pool obtained during step 9 by performing the amplification (e.g., the PCR) under mutagenic conditions. This mutagenic step broadens the sequence diversity of the pool which is subjected to the selection.
- the in vitro selection of the aptazyme generally yields a set of several sequences, which exhibit the desired property of ligand-dependent self-cleavage. It is then possible to clone the selected aptazymes and determine their exact sequence. This can be achieved by ligation of the double-stranded DNA pool encoding the aptazymes to an appropriate linearized plasmid vector and transforming bacteria with the resulting circular plasmid vectors (Sambrook et al (1989). Molecular cloning, a laboratory manual, Second Edition, N. Ford, ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press). The cloned plasmids containing the aptazymes can be sequenced by the method of Sanger et al.
- reporter genes expression cassettes and vectors carrying them can be constructed by standard molecular biology techniques (Sambrook et al (1989). Molecular cloning, a laboratory manual, Second Edition, N. Ford, ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press).
- the target cells are transfected with two reporter genes; reporter gene (1) contains the sequence of an in v/tro-selected aptazyme within at least one UTR, and reporter gene (2) does not contain any aptazyme. Two sets of cells are cultured in the absence and in the presence of the ligand.
- the ratio of reporter gene (1) expression over reporter gene (2) expression is assessed in the presence and in the absence of ligand.
- the aptazyme clone which yields the lowest ratio where protein production is to be repressed and the highest ratio where protein production is to be induced is selected.
- the invention can be used to control protein production in vitro, in vivo or ex vivo in various cell types.
- the invention also resides in a method for controlling production of a protein, polypeptide or peptide from a gene, comprising inserting, within at least one untranslated region of the gene, a nucleic acid sequence conferring ligand-dependent self- cleavage to a RNA transcribed from said gene.
- the invention provides a method for removing, in a ligand-dependant manner, the 5 '-cap structure and/or the 3'-polyA tail from a pre-mRNA or a mRNA of a gene whose expression is to be controlled. This can be achieved by inserting a sequence in at least one UTR of the gene which entails ligand-dependent self- cleavage of the transcribed RNA.
- the invention thus also resides in a method of removing a 5 '-cap structure and/or a 3'-polyA tail from a pre-mRNA or a mRNA transcribed from a gene, comprising inserting, within at least one untranslated region of the gene, a nucleic acid sequence conferring ligand-dependent self-cleavage to a RNA transcribed from said gene.
- the method is for removing a 5 '-cap structure from a pre-mRNA or a mRNA transcribed from a gene, and the nucleic acid sequence is inserted within at least a 5 '-untranslated region of the gene.
- the method is for removing a 3'-polyA structure from a pre-mRNA or a mRNA transcribed from a gene, and the nucleic acid sequence is inserted within at least a 3 '-untranslated region of the gene.
- the invention also relates to a method of controlled production of a protein, polypeptide or peptide within a cell, comprising (i) introducing into a cell a modified gene encoding the protein, polypeptide or peptide, wherein said modified gene contains, inserted in an untranslated region thereof (UTR), a nucleic acid sequence conferring ligand-dependant self-cleavage to a RNA molecule transcribed from the gene, and (ii) contacting the cell in the presence or absence of the ligand.
- UTR untranslated region thereof
- the modified gene contains, inserted in an untranslated region thereof (UTR), a nucleic acid sequence conferring ligand-activated self-cleavage to a RNA molecule transcribed from the gene, and production is repressed by contacting the cell with the ligand, while production is restored (or increased) by removing (or stopping contacting or in the absence of) the ligand.
- UTR untranslated region thereof
- the modified gene contains, inserted in an untranslated region thereof (UTR), a nucleic acid sequence conferring ligand-inhibited self-cleavage to a RNA molecule transcribed from the gene, and production is increased by contacting the cell with the ligand, while production is depressed (or inhibited) by removing (or stopping contacting or in the absence of) the ligand.
- UTR untranslated region thereof
- the ligand can be either exogenously supplied to the cell or be an endogenous component of the cell, as described above.
- the ligand can be a molecule which is preferentially present in cells where production of the protein, polypeptide or peptide is sought (and essentially absent or express at a lower concentration in other cells or tissues).
- the above method can be used for production of a protein, polypeptide or peptide in a cell (or in a cell population or culture) in vitro or ex vivo.
- the above method can also be applied to the production of a protein, polypeptide or peptide in a cell, tissue or organ in vivo.
- the above cell can be selected from prokaryotic cells, eukaryotic cells, mammalian cells and plant cells, for instance. It may be a tissue, organ, an isolated cell culture, established cell lines or primary cultures. Preferred cells are mammalian cells, in particular murine or human cells, more preferably ex vivo or in vivo.
- the gene can be introduced into the cells by administration to an organism, and the ligand can be supplied exogenously or contained within the cell.
- the gene may be contained in a vector selected from plasmids, viruses, cosmids, artificial chromosomes, etc., or combinations thereof.
- Preferred vectors include plasmids and viruses, such as adenoviruses, retroviruses, AAV, HSV, HIV, etc.
- the instant invention also discloses and claims combinations of a ligand and a modified gene (or vector containing the same) as described above, for simultaneous, separate or sequential use.
- Preferred combinations comprise:
- RNA transcribed from the said gene is activated by said ligand.
- the ligand is selected from the group of nucleic acid molecules, proteins, polysaccharides, sugars and organic and inorganic molecules.
- RNA sequence e.g., a ribozyme hammerhead sequence
- Figure 2A reporter protein production without ribozyme sequence, with inactive ribozyme sequence or with active ribozyme sequence in one UTR of the gene.
- Figure 2B site of insertion of the ribozyme sequences (restriction enzymes sites) within the pre-mRNA of the reporter gene.
- Figure 2C sequence of the active and the inactive ribozyme.
- Figure 3A outline of the selection procedure for ligand-inhibited self-cleaving RNA sequences.
- Figure 3B outline of the selection procedure for ligand-activated self-cleaving RNA sequences.
- Figure 6 Time course of the two selections. The percentage of RNA eluted after each selection cycle is shown for the two selections, using doxycycline or pefloxacine as a ligand. An empty space between two cycles indicates an increase in the selection pressure (either by changing incubation times or/and by decreasing ligand concentration (see Table 1).
- Figure 7 shows that
- RNA was incubated in 50mM Tris-HCl pH7.5 at 37°C, self-cleavage was initiated by addition of lOmM MgCl 2 and assessed during 3 minutes. When indicated 200nM of doxycycline (Dox) or tetracycline (Tet) were added in the cleavage reaction.
- Dox doxycycline
- Tet tetracycline
- the self-cleavage reaction was performed in the absence of added RNA (left panel: Control), in the presence of l ⁇ M and 2 ⁇ M aptazyme-derived aptamer (2 nd and 3 rd panels from the left: l ⁇ M aptamer, 2 ⁇ M aptamer), in the presence of l ⁇ M and 2 ⁇ M non-cognate RNA (4 th and 5 th panels from the left: l ⁇ M non-cognate RNA, 2 ⁇ M non-cognate RNA).
- Example 1 Selection of aptazymes inhibited by doxycycline and pefloxacine
- the initial degenerated pool was produced using standard DNA synthesis chemistry on an Expedite nucleic acids synthesis system (Millipore). Oligonucleotides were purified on denaturating polyacrylamide gels. The following primers were synthesized: Pool-Primer:
- This DNA library was used as a template for T 7 RNA polymerase during the initial in vitro selection cycle.
- Standard transcription reactions contained the following components: 250 units T 7 RNA polymerase (Stratagene), T 7 Buffer (Stratagene), 2,5 mM of each dNTP, 20 mM of GMPS, 20 ⁇ Ci ⁇ 32 P GTP, RNasin (50 U), ligand (ImM).
- the transcription reaction was incubated over night at 37°C.
- the uncleaved RNA transcripts were then purified on a 8% denaturating polyacrylamide gel. After purification, the RNA was treated with Iodo- Acetyl-LC-Biotin (Pierce - 200 fold excess) for 90 min. at room temperature.
- RNA was gel purified under the same conditions as described above and incubated with streptavidine agarose (Pierce) for 30 min. at room temperature. The column material was then washed 6 times alternatively in 1ml washing buffer A (WA ; 25 mM HEPES pH 7.4, 1M NaCl, 5mM EDTA), washing buffer B (WB ; 3M urea, 5mM EDTA) and water. Finally, it was rinsed five times with water.
- washing buffer A WA ; 25 mM HEPES pH 7.4, 1M NaCl, 5mM EDTA
- washing buffer B washing buffer B
- WB 3M urea, 5mM EDTA
- the immobilized RNA was incubated at 37°C for 2 to 4 hours in selection buffer (SB ; 40 mM Tris HCl pH 8, 50 mM NaCl, 10 mM Spermidine, 8 mM MgCl 2 ) in the presence of the ligand (1 mM to 1 ⁇ M).
- the incubation was started upon the addition of magnesium. After round 5, this incubation was interrupted 10 times during the first 2h30 of incubation and the column was washed twice with 1 ml WB (denaturating conditions) and rinsed 3 times with water. The column material was then washed again thoroughly (6 times 1 ml WA - WB - H 2 O; 5 times 1 ml H 2 O).
- RNA was finally reverse transcribed with Tth DNA polymerase (Boehringer) and amplified by PCR using primers 3 and 4 under standard conditions. This DNA template was used as a template for T 7 RNA polymerase for the next round of selection.
- RNA quantity was reduced.
- the ligand concentration, and the different incubation times were also adjusted to increase the stringency.
- the pool was subjected to a mutagenic PCR under previously described conditions (Cadwell R.C. & Joyce G.F.; in PCR Methods and Applications (1992): 28-33).
- the mutagenic PCR creates a new diversity of RNA molecules closely related to selected sequences.
- the selection scheme was modified by introduction of several denaturation steps during the negative selection (see ii-b).
- Radio-labeled RNA molecules were gel purified and incubated at a 10 nM concentration in SB with various amounts of ligand at 37°C. Reaction was initiated upon addition of magnesium. Aliquots were taken at different time points and mixed with an equal volume of loading buffer (9M urea, 5 mM EDTA) on ice to quench the reaction. The samples were then loaded on a 12 % sequencing gel, and the bands corresponding to intact (103 nt) or cleaved (13 nt) nucleic acid sequences were quantified using a phosphor imager (Molecular Dynamics).
- Example 2 Selection of doxycycline- and pefloxacine-induced expression cassettes in mammalian cells
- the DNA pools obtained after selection cycles 10, 13 and 16 are cut by the restriction enzymes Xhol and Kpnl.
- the plasmids pNPGl and pNPG2 (figure 4) are cut with the same enzymes and the three DNA pools are ligated to each of the two plasmids.
- E. Coli strain DH5 ⁇ is transfected by each one of the six ligation mixture according to standard procedures (Sambrook et al (1989). Molecular cloning, a laboratory manual, Second Edition, N. Ford, ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press). One hundred colonies are isolated for each transfection. Plasmid DNA is extracted and purified from each of these colonies.
- the aptazymes cloned into the plasmids are sequenced by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74 (1977) 5463- 5467) with an Applied Biosystems kit according to the manufacturer's instructions.
- Human cultured cell lines HeLa and HEK293 are transfected by plasmid DNA complexed to lipofectamine (Gibco-Life Sciences, Bethesda, MD USA) according to the manufacturer's instructions (6 ⁇ l lipofectamine per ⁇ g DNA). In one set of experiments the cells are incubated with 10 ⁇ M doycycline or 10 ⁇ M pefloxacine, in another set of experiment the cells are incubated without ligand. 48 hours after transfection, the cells are lysed and the amount of Renilla luciferase and firefly lucifease are recorded according to the manufacturer's instructions (Promega, Madison, Wl USA).
- the ratio of firefly luciferase activity (aptazme-controlled) over Renilla luciferase activity (Ratio R) is computed for each experimental condition (i.e. plasmid, ligand).
- the plasmids which yield production of high amount of firefly luciferase in the presence of ligand (doxycycline or pefloxacine) and low amount of firefly luciferase in the absence of ligand are identified by the highest ratio R. These plasmids are selected for further use in gene transfer experiments where production of a protein is to be controlled by doxycycline or pefloxacine.
- Example 3 Doxycycline- induced protein production in mice 10 ⁇ g of aptazyme-containing plasmids pNPGl or pNPG2 (selected as in example 2) are injected into tibialis cranialis muscles (posterior limbs) of eight weeks-old Balb/C mice, the limbs are subsequently subjected to electroporation in order to enhance gene transfer (Mir, L.M. et al.; P.N.A.S. USA (1999); 96(8):4262-7).
- One group of five mice is given doxycycline in the drinking water (0.2 mg ml), the other group is not. 7 days after gene transfer the animals are sacrificed and the tibialis cranialis muscles are collected.
- the muscles are resuspended in 1ml of lysis buffer (Dual-Luciferase reporter Assay System, Promega, Madison, Wl USA) homogenized by glass and ceramic beads into a Fast-Prep homogenizer (Bio 101, Vista, CA USA).
- the amount of firefly luciferase and Renilla luciferase are assessed according to Promega' s instructions.
- the amount of firefly luciferase synthesized by the mice of each group (treated or not by doxycycline) is compared. Inter-individual variations in gene transfer efficiency can be compensated by comparing the ratio firefly luciferase over renilla luciferase between the 2 groups of mice. Hence one can verify that luciferase production in mice is induced by doxycycline.
- Example 4 selection of RNA aptamers binding to doxycycline ( Figure 8) Aptazymes were selected according to the procedure outlined in example 1. The DNA pool was inserted into pNPG2 plasmid according to example 2. Several clones were sequenced by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74 (1977) 5463- 5467) with an Applied Biosystems kit according to the manufacturer's instructions.
- the amplified DNA sequence (SEQ ID NO: 5) was transcribed by T7 RNA polymerase and the resulting RNA was purified as described in example 1.
- the RNA sequence is (hereafter referred to as the aptazyme):
- Primer 4 The clone 6 DNA sequence was also amplified by PCR using primer 4 and primer 3. Primer 4 and the amplified sequence (upper strand) are described below: Primer 4:
- Twice-underlined T7 RNA polymerase transcription promoter Underlined: random sequence in the original DNA pool (example 1)
- Bold mutated base Primer 4 introduces a C to G mutation which inactivates the self-cleavage activity of hammerhead ribozymes but minimally affects their tertiary structure (Baidya, N. and Uhlenbeck, O.C.; Biochemistry (1997) 36: 1108-1114).
- the amplified DNA sequence (SEQ ID NO: 7) was transcribed by T7 RNA polymerase and the resulting RNA was purified as described in example 1.
- the RNA sequence is (hereafter referred to as the aptamer):
- the experiment described in figure 8 shows 1) that the aptazyme, features self- cleavage activity which is inhibited specifically by doxycycline (tetracycline, an isomer of doxycycline, inhibits cleavage to a much lesser extent) and 2) that the aptamer is able to relieve inhibition of self-cleavage by doxycycline but not inhibition of self-cleavage by tetracycline.
- the interpretation is that the aptamer competes out the aptazyme for the binding of doxycycline. This is a demonstration that mutation of the nucleotide C 5' to the cleavage site into nucleotide G, converts the aptazyme into a RNA ligand which binds doxycycline specifically.
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Application Number | Priority Date | Filing Date | Title |
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EP00977426A EP1224305A2 (de) | 1999-10-15 | 2000-10-13 | Selbstschneidende rna-sequenzen und deren verwendung in der kontrole der proteinsynthese |
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EP99402552 | 1999-10-15 | ||
EP99402552A EP1092777A1 (de) | 1999-10-15 | 1999-10-15 | Selbst-schneidende RNA-Sequenzen und deren Verwendung zur Kontrolle der Protein-Synthese |
PCT/EP2000/010423 WO2001029234A2 (en) | 1999-10-15 | 2000-10-13 | Self-cleaving rna sequences and their use for the control of protein synthesis |
EP00977426A EP1224305A2 (de) | 1999-10-15 | 2000-10-13 | Selbstschneidende rna-sequenzen und deren verwendung in der kontrole der proteinsynthese |
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EP99402552A Withdrawn EP1092777A1 (de) | 1999-10-15 | 1999-10-15 | Selbst-schneidende RNA-Sequenzen und deren Verwendung zur Kontrolle der Protein-Synthese |
EP00977426A Withdrawn EP1224305A2 (de) | 1999-10-15 | 2000-10-13 | Selbstschneidende rna-sequenzen und deren verwendung in der kontrole der proteinsynthese |
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KR (1) | KR20020037384A (de) |
CN (1) | CN1377414A (de) |
AU (1) | AU784832B2 (de) |
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MX (1) | MXPA02002864A (de) |
NO (1) | NO20021713L (de) |
NZ (1) | NZ518396A (de) |
PL (1) | PL355165A1 (de) |
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US6858390B2 (en) | 1998-12-31 | 2005-02-22 | Ingeneus Corporation | Aptamers containing sequences of nucleic acid or nucleic acid analogues bound homologously, or in novel complexes |
CN108350451A (zh) * | 2015-11-12 | 2018-07-31 | 贝勒医学院 | 通过适体介导的多腺苷酸化调节来外源性控制哺乳动物基因表达 |
JP2022507269A (ja) * | 2018-11-13 | 2022-01-18 | オンコラス, インコーポレイテッド | カプセル化ポリヌクレオチド及び使用方法 |
CN116555253A (zh) * | 2022-01-30 | 2023-08-08 | 中国科学院分子细胞科学卓越创新中心 | 含高均一性poly(A)尾的mRNA及其制备方法 |
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AU679525B2 (en) * | 1992-12-04 | 1997-07-03 | Innovir Laboratories, Inc. | Regulatable nucleic acid therapeutic and methods of use thereof |
US5663064A (en) * | 1995-01-13 | 1997-09-02 | University Of Vermont | Ribozymes with RNA protein binding site |
WO1998043993A2 (en) * | 1997-03-31 | 1998-10-08 | Yale University | Nucleic acid catalysts |
WO1999018116A1 (en) * | 1997-10-07 | 1999-04-15 | Smithkline Beecham Corporation | Method for modulating gene expression |
AU6522599A (en) * | 1998-10-23 | 2000-05-15 | Children's Medical Center Corporation | Use of a self-cleaving rna motif to modulate gene expression |
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CA2387185A1 (en) | 2001-04-26 |
NZ518396A (en) | 2004-01-30 |
WO2001029234A2 (en) | 2001-04-26 |
HUP0203268A2 (hu) | 2003-01-28 |
JP2003512059A (ja) | 2003-04-02 |
CZ20021313A3 (cs) | 2002-07-17 |
HUP0203268A3 (en) | 2005-08-29 |
NO20021713D0 (no) | 2002-04-11 |
KR20020037384A (ko) | 2002-05-18 |
ZA200202823B (en) | 2003-09-23 |
BR0014632A (pt) | 2002-06-18 |
EP1092777A1 (de) | 2001-04-18 |
NO20021713L (no) | 2002-06-12 |
MXPA02002864A (es) | 2003-07-21 |
AU1514901A (en) | 2001-04-30 |
CN1377414A (zh) | 2002-10-30 |
WO2001029234A3 (en) | 2001-11-29 |
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PL355165A1 (en) | 2004-04-05 |
AU784832B2 (en) | 2006-07-06 |
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