WO1999027135A2 - Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules - Google Patents

Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules Download PDF

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WO1999027135A2
WO1999027135A2 PCT/US1998/024854 US9824854W WO9927135A2 WO 1999027135 A2 WO1999027135 A2 WO 1999027135A2 US 9824854 W US9824854 W US 9824854W WO 9927135 A2 WO9927135 A2 WO 9927135A2
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cells
rna
gene
egs
molecule
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PCT/US1998/024854
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WO1999027135A3 (fr
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Timothy W. Nilsen
Hugh D. Robertson
Thomas J. Kindt
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Yale University
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Priority claimed from US08/976,220 external-priority patent/US6013447A/en
Application filed by Yale University filed Critical Yale University
Priority to CA002310510A priority Critical patent/CA2310510C/fr
Priority to JP2000522276A priority patent/JP2001524317A/ja
Priority to AU15323/99A priority patent/AU732321B2/en
Priority to EP98959542A priority patent/EP1032707A2/fr
Publication of WO1999027135A2 publication Critical patent/WO1999027135A2/fr
Publication of WO1999027135A3 publication Critical patent/WO1999027135A3/fr

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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/126—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes involving RNAse P

Definitions

  • RNAs biologically active nucleic acids
  • a major challenge involves the functional analysis of the available and forthcoming genomic information; i.e. determination of the biological role of genes revealed by sequencing. It is particularly important to identify those genes that encode proteins essential for viability. Such proteins are of clear significance in the development of effective chemotherapeutic agents targeted to pathogenic organisms.
  • functional genomic analysis including bioinformatics, expression analysis, and targeted gene disruption. Informatics alone is unlikely to provide definitive new insight into gene function. For example, although E. coli is the best studied organism by far, the genomic sequence revealed that approximately forty percent of the genes were of unknown function.
  • Expression profiling provides primarily inferential information, and targeted gene disruption, although definitive, is labor intensive and time consuming.
  • Ribonucleic acid (RNA) molecules can serve not only as carriers of genetic information, for example, genomic retroviral RNA and messenger RNA (mRNA) molecules and as structures essential for protein synthesis, for example, transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, but also as enzymes which specifically cleave nucleic acid molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • Such catalytic RNA molecules are called ribozymes.
  • ribozymes theoretically can cleave any desired site in an RNA molecule, in reality not all sites are efficiently cleaved by ribozymes designed to cleave them. This is especially true in vivo where numerous examples have been described of sites that are inefficiently cleaved by targeted ribozymes.
  • Kawasaki et al Nucl. Acids Res. 24(15):3010-3016 (1996), describes the use of a transcript encoding a fusion between adenovirus El A- associated 300 kDa protein (p300) and luciferase to assess the efficiency with which sites in the p300 RNA are cleaved by hammerhead ribozymes in vivo.
  • a few hammerhead ribozymes targeted to sites having GUX triplets (which are required for cleavage by a hammerhead ribozyme) were designed and expressed from a vector in cells.
  • a separate vector expressed the p300-luciferase fusion RNA. Cleavage of sites in the p300 portion of the transcript was assessed by measuring luciferase activity.
  • Kawasaki et al. tested each ribozyme separately and therefore their method also does not solve the need for a rapid, efficient selection process.
  • RNA molecules cannot be accurately predicted from theoretical considerations and the determination of actual secondary and tertiary structure of an RNA molecule requires extensive experimentation. It can also be difficult to identify ribozymes and other biologically active molecules that will function inside cells since not all such biologically active molecules that are functional in vitro are functional in cells because they are, for example, improperly localized, sequestered, or bound by intracellular proteins.
  • RNA molecules such as ribozymes, EGSs for ribozymes, and antisense RNA, that alter expression of an RNA molecule efficiently in vivo.
  • the first provides a means for rapidly and efficiently identifying essential and functional genes; and the second provides a means for obtaining biologically active nucleotide molecules (ribozymes, EGSs, and antisense), which can be used inactivate functional genes.
  • biologically active nucleotide molecules ribozymes, EGSs, and antisense
  • a library of EGSs is prepared based on all possible known compositions. This is readily calculated knowing the minimum sequence requirements required for targeting and cleavage by RNase P and the length of the EGS based on the predicted size of the genome to be screened.
  • the EGSs are twelve or thirteen-mers for targeting bacterial RNase P to cleave a substrate.
  • This library of EGSs is added to the cells containing the genes to be screened, for example, E. coli. Additional methods may be used to amplify the library, or the cells which survive exposure to the EGSs. Those cells in which the EGS causes a loss of viability, or other phenotype, are identified. The EGS(s) responsible for the loss of viability are analyzed, and the resulting sequence information used to identify the gene within the known genomic sequences.
  • nucleotide molecules with optimal biological activity are rapidly identified through the use of a vector including two reporter genes, the first in frame with the gene of interest, and the second as a control to verify that the vector is present in a cell or to aid in selection of cells containing the vector.
  • the vector may include a gene encoding any protein that confers drug resistance in a bacteria (the gene of interest) in frame with a beta-galactosidase gene (reporter gene 1) and a gene encoding antibiotic resistance (reporter gene 2).
  • the vector also includes one of many possible functional oligonucleotide molecules (such as an EGS), although this can also be provided on a separate vector.
  • the vector(s) is added to cells such as E. coli.
  • the cells containing the vectors can be isolated by treatment with the antibiotic that kills all the cells that do not express the gene for antibiotic resistance.
  • Those cells where the gene of interest is cleaved by the functional oligonucleotide molecule can then be identified by reference to reporter gene 1.
  • Those cells which are identified can then be amplified.
  • the gene of interest is essential for viability. In this case, the plate with the bacteria is first replicated, then the cells which are killed by cleavage of the mRNA of interest are identified, and the responsible functional oligonucleotide molecules isolated from the duplicate plate.
  • Figure 1 is a diagram of the mechanism of action of EGSs on cleavage of target mRNAs.
  • Figure 2a is a diagram of the constructs used in the examples to make an ARA-Nl 1 library (13-mer EGSs, NnCCACCA), for use in identifmg essential genes in E. coli.
  • Figure 2b is a schematic of the pARANx (A K) EGS transcription vector.
  • Figure 2C is a schematic of the pARAN vector and library construction.
  • Figure 3 is a flow diagram of the method for induction and selection on solid media of the AraNl 1 library in E. coli. Only cells expressing an appropriate EGS survive and are amplified.
  • Figure 4 is a flow diagram of the ampicillin enrichment method for selection of EGS targeting essential genes.
  • Figures 5 a and 5b are graphs of the growth of ampicillin selected clone E8-1 ( Figure 5 a) and growth of control ( Figure 5b) with (circles) and without (squares) arabinose.
  • Figure 6 is a graph of the relative numbers of active EGSs identified in libraries of different size EGSs: 9-mers, 10-mers, 11-mers, and 12-mers.
  • Figure 7 is a diagram of an example of a vector for use in the method for identifying functional oligonucleotide molecules including EGS, ribozymes, and antisense.
  • Reporter gene 1 encodes a fusion transcript made up of an RNA of interest and RNA encoding a reporter protein (reporter protein A).
  • the fusion transcript encodes a fusion protein made up of the protein encoded by the RNA of interest and reporter protein A.
  • Reporter gene 2 encodes reporter protein B.
  • the targeting gene encodes one of the functional oligonucleotide molecules to be tested.
  • Figure 8 is a diagram of an example of a vector for use in the method to identify functional oligonucleotide molecules.
  • Reporter gene 1 encodes a fusion transcript made up of an RNA encoding chloramphenicol acetyltransferase (CAT) and RNA encoding ⁇ -galactosidase (reporter protein A).
  • the fusion transcript encodes a fusion protein made up of CAT and ⁇ -galactosidase.
  • Reporter gene 2 is an ampicillin resistance gene.
  • the targeting gene is an EGS cassette encoding one of a library of 50 EGS molecules, each targeted to a different site in the CAT RNA.
  • Figures 9a and 9b are graphs of cell culture density (A 6 oo) versus time (in minutes) of cells in the presence of 5 ⁇ g/ml chloramphenicol ( Figure 9a) or 25 ⁇ g/ml chloramphenicol ( Figure 9b).
  • the cells contained a vector similar to the vector shown in Figure 8 that did not encode an EGS (circles), encoded EGS 36 (triangles), encoded EGS 20 (inverted triangles), or encoded both EGS 52 (diamonds).
  • Figure 10 is a graph of the percent inhibition of chloramphenicol acetyl transferase (CAT) activity by EGS-CAT-1, EGS-CAT-2, EGS-20, EGS-31, EGS-36, and EGS-52.
  • Each X represents an EGS complementary to a specific sequence of the mRNA transcript of the CAT gene. The X's show that EGSs were recovered and that the library was represented in the experiment. The circles indicate the relative efficiency by which each EGS knocks down the expression of the targeted gene.
  • the functional oligonucleotide molecules are EGSs and the desired RNA molecule is the RNA transcribed from a known gene.
  • RNA molecules can serve not only as carriers of genetic information, for example, genomic retroviral RNA and messenger RNA (mRNA) molecules and as structures essential for protein synthesis, for example, transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, but also as enzymes which specifically cleave nucleic acid molecules or as elements which direct an enzyme which specifically cleaves nucleic acid molecules. Any of these RNAs can be a target for cleavage or inactivation by a functional oligonucleotide molecule.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • a key advantage of the disclosed methods and vectors is the assessment of alteration of expression of an RNA of interest in an in vivo setting which will be the same or similar to the setting where identified functional oligonucleotide molecules, or affector oligomers based on such identified RNA molecules, will be used.
  • Another advantage of the disclosed methods is that all, or a substantial number, of the accessible sites in the RNA of interest can be determined in one assay. Such sites, determined to be accessible for one type of functional oligonucleotide molecule, may be accessible for other types of functional oligonucleotide molecules.
  • the disclosed methods allow assessment not just of cleavage of the RNA of interest, but also of an ultimate desired phenotype (that is, loss of the phenotype supported by the RNA of interest) as a result of such cleavage.
  • the RNA molecule of interest can be any RNA molecule or portion of an RNA molecule that can be transcribed. It is preferred that the RNA molecule of interest be an RNA molecule involved in the expression of a gene of interest, the expression of which is to be inhibited.
  • the RNA molecule can be a mRNA, a portion of a mRNA, a pre-mRNA including introns, or an intron. Alternatively the RNA molecule can be a viral RNA.
  • Important pathogens include the bacteria Pseudomonas aeruginosa, Mycobacterium tuberculosis, Hemophilus influenzae, Staphylococcus aureus, My coplasma pneumoniae, Escherichia coli, Streptococcus pneumoniae, Neisseria gonorrhaoeae, Streptococcus viridans, Streptococcus pyogenes, Proteus mirabilis, Proteus vulgaris, Salmonella typhimurium, Shigella dysentereae, Clostridium difficile, and Klebsiella pneumoniae, and the fungi Candida albicans, Aspergillus flavus, Aspergillus fumagatus, and Histoplasmatus capsulatum.
  • RNA molecules are designed to alter, or preferably inhibit, the expression of an RNA of interest. These molecules can be ribozymes, EGSs for RNase P, or antisense RNA. Ribozymes and EGSs inhibit expression of an RNA molecule by cleaving or mediating cleavage of the RNA molecule at a targeted site. Antisense RNA or DNA inhibits expression of an RNA molecule through a sequence-specific interaction with the RNA molecule. External Guide Sequences (“EGSs ”)
  • RNA sequence in a prokaryotic or eukaryotic cells can be converted into a substrate for RNase P.
  • the substrate is created using an EGS having at its 5' terminus nucleotides complementary to the nucleotides 3' to the cleavage site in the RNA to be cleaved and at its 3' terminus the nucleotides NCCA (N is any nucleotide). This is described in U.S. Patent No. 5,168,053, WO 92/03566 and Forster and Altman, Science 238:407-409 (1990).
  • EGS for promoting RNase P-mediated cleavage of RNA has also been developed for use in eukaryotic systems as described by U.S. Patent No. 5,624,824, Yuan et al, Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992), WO 93/22434, WO 95/24489, and WO 96/21731.
  • "external guide sequence” and “EGS” refer to any oligonucleotide or oligonucleotide analog that forms, in combination with a target RNA, a substrate for RNase P. EGS technology has been used successfully to decrease levels of gene expression in both bacteria (Altman et al.
  • the requirements for an EGS functional with prokaryotic RNase P are less stringent than those for a eukaryotic EGS.
  • the critical elements of a prokaryotic EGS are (1) nucleotide sequence which specifically binds to the targeted RNA substrate to produce a short sequence of base pairs 3' to the cleavage site on the substrate RNA and (2) a terminal 3'-NCCA, where N is any nucleotide, preferably a purine.
  • the sequence generally has no fewer than four, and more usually six to fifteen, nucleotides complementary to the targeted RNA. It is not critical that all nucleotides be complementary.
  • the rate of cleavage is dependent on the RNase P, the secondary structure of the hybrid substrate, which includes the targeted RNA and the presence of the 3'-NCCA in the hybrid substrate.
  • Eukaryotic EGSs also promote cleavage by prokaryotic RNase P and can be used for this purpose.
  • An EGS for promoting cleavage by eukaryotic RNase P contains sequences which are complementary to the target RNA and which forms secondary and tertiary structure akin to portions of a tRNA molecule.
  • a preferred form of eukaryotic EGS contains at least seven nucleotides which base pair with the target sequence 3' to the intended cleavage site to form a structure like the amino acyl acceptor stem (A stem), nucleotides which base pair to form a stem and loop structure similar to the T stem and loop, followed by at least three nucleotides that base pair with the target sequence to form a structure like the dihydroxyuracil stem.
  • SEGS Short External Guide Sequence
  • Ribozymes include any trans-cleaving catalytic nucleic acid.
  • Several classes of such ribozymes are known and have been either adapted or designed to cleave RNA molecules in a site-specific manner.
  • Intron-derived ribozymes are derived from self-excising introns found in Tetrahymena RNA, as described in U.S. Patent No. 4,987,071, WO 88/04300, and Cech, / ⁇ / ⁇ . Rev. Biochem. 59:543-568 (1990).
  • Hammerhead ribozymes are derived from self-cleaving RNA molecules present in certain viruses (Buzayan et al, Proc. Natl. Acad. Sci.
  • hammerhead ribozymes for the specific cleavage of target RNA molecules and their use is described in U.S. Patent No. 5,254,678, WO 89/05852, EP 321021, and U.S. Patent No. 5,334,711. Derivatives of hammerhead ribozymes are described in U.S. Patent No. 5,334,711; WO 94/13789; and WO 97/18312. Axehead ribozymes are derived from self- cleaving domains in some viroid RNAs such as hepatitis delta virus (U.S. Patent No.
  • Antisense molecules are usually single stranded DNA or RNA molecules, or their substituted analogues, which can bind to the target RNA through Watson and Crick base pairing and prevent the translation of these RNAs (Stein CA, Antisense Nucleic Acid Drug Dev 8(2): 129-32 (1998); Crooke S ', Antisense Nucleic Acid DrugDev 8(2): 115-22 (1998); Akhtar S, J Drug Target. 5(4):225-34 (1998); Mizuno, T., et al, Proc. Natl. Acad. Sci.
  • DNA based antisense can also inhibit expression of proteins by presenting the DNA-RNA hybrid as a target for cleavage by the endogenous RNaseH enzyme (Crooke ST, Antisense Nucleic Acid DrugDev 8(2):133-4 (1998); Caselmann et al., Intervirology 40(5-6):394-9 (1997); Giles, R.V. and Tidd, D.M., Nucleic Acid Res., 20, 763 (1992)), thereby destroying the target RNA.
  • the antisense molecules can be made more resistant to nucleases by introducing chemical modifications, such as 2' modifications and phosphorothioate diester linkages instead of the phosphodiester linkage (Agrawal S and Zhao Q, Antisense Nucleic Acid DrugDev 8(2):135-9 (1998); Agrawal, S., et al, Proc. Natl. Acad. Sci., USA, 85, 7089, (1988)) and duplexes of these molecules with an RNA is recognized by RNaseH.
  • the disclosed methods use vectors which have certain elements in common, including reporter genes, genes for selection of cells which do, or do not, contain the functional oligonucleotide molecules, and the necessary sequences for expression and replication of the vectors.
  • the disclosed vectors are generally of two forms, with each form adapted to either the assay for identifying essential and functional genes or the assay to identify functional oligonucleotide molecules. These are generally described as follows.
  • Vectors for Use in Identifying Essential or Functional Genes encode a functional oligonucleotide molecule including a degenerate targeting sequence.
  • the set of encoded functional oligonucleotide molecules include functional oligonucleotide molecules targeted to every possible sequence.
  • the length of the targeting sequence is preferably chosen to match the length of unique sequences present in the genome of cells to be assayed. This relationship is described in more detail below. The effect is to obtain a set of vectors that collectively encode a set of functional oligonucleotide molecules targeted to every possible unique sequence in the genome of the cells of interest.
  • the set of vectors is transformed or transfected into the cells, and the cells screened or selected for cell death or for a change in a phenotype of interest.
  • the selected cells harbor the functional oligonucleotide molecules which are targeted to essential or functional genes in the cells.
  • the functional oligonucleotide molecules can then be identified by characterizing the vectors in the cells.
  • the genes targeted by the functional oligonucleotide molecules can be identified by correlating the targeting sequences in the functional oligonucleotide molecules with the known genomic sequences.
  • Vectors for use in functional oligonucleotide molecule assays include a reporter gene 1 encoding the fusion transcript including the RNA molecule of interest and RNA encoding reporter protein A. Inactivation of the RNA molecule of interest alters expression of the reporter protein A.
  • the vectors also include a second reporter gene 2 encoding a second reporter protein B. Expression of the second reporter protein B can be used both to detect transformation or transfection of the vector into cells and as a control for effects on the expression of the first reporter protein that are not due to inhibition of expression of the RNA molecule of interest.
  • the vector also encodes a functional oligonucleotide molecule targeted to the RNA of interest.
  • the method preferably uses a set of these vectors where each vector in the set encodes a different functional oligonucleotide molecule, each targeted to a different site in the RNA molecule of interest.
  • the set of vectors is transformed or transfected into appropriate cells, and the cells are screened or selected for expression of the second reporter protein B.
  • the cells expressing reporter protein B are then screened or selected for those cells which do not express the first reporter protein A, or express reporter protein A only at a low level. These cells harbor the most efficient functional oligonucleotide molecules which then can be identified by characterizing the vectors in the cells.
  • the vectors can be autonomously replicating vectors, viral vectors, nucleic acids that integrate into the host chromosome, and transiently expressed nucleic acid molecules.
  • the reporter genes can be expressed using any suitable expression sequences. Numerous expression sequences are known and can be used for expression of the reporter genes. Expression sequences can generally be classified as promoters, terminators, and, for use in eukaryotic cells, enhancers. Expression in prokaryotic cells also requires a Shine-Dalgarno sequence just upstream of the coding region for proper translation initiation. Inducible promoters are preferred for use with the first reporter gene since it is preferred that expression of the first reporter gene be adjustable.
  • plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell be used with these hosts. It is preferred that the vector carry a replication sequence.
  • the vectors can be used to transiently transfect or transform host cells, or can be integrated into the host cell chromosome.
  • the vectors include sequences that allow replication of the vector and stable or semi-stable maintenance of the vector in the host cell. Many such sequences for use in various eukaryotic and prokaryotic cells are known and their use in vectors routine. Generally, it is preferred that replication sequences known to function in host cells of interest be used.
  • origins of replication from vectors such as pBR322 and pUC19 are preferred for prokaryotic cells
  • origins of replication from such vectors as YEP24 and YRP17 are preferred for fungal cells
  • origins of replication from SV40 and pEGFP-N are preferred for eukaryotic cells. All of these examples are commercially available (New England Biolabs; Clontech).
  • a preferred vector for use in prokaryotic cells is Bluescript-SK + (Stratagene).
  • a preferred vector for use in eukaryotic cells is the shuttle vector pEGFP-N (Clontech). This vector encodes a green fluorescent protein (GFP) that has been optimized for maximal activity in mammalian cells and is designed for expression of GFP fusion proteins.
  • This vector also contains a multiple cloning site (MCS) 5' to the GFP sequence which is designed for creating fusion proteins in all three reading frames.
  • the MCS can be used for inserting DNA encoding an RNA of interest to generate a gene encoding a fusion transcript which encodes a fusion protein.
  • Reporter proteins can be any proteins which can be detected either directly or indirectly.
  • GFP green fluorescent protein
  • Reporter proteins producing a fluorescent signal are useful since such a signal allows cells to be sorted using FACS.
  • Another way of sorting cells based on expression of the reporter protein involves using the reporter protein as a hook to bind cells.
  • a cell surface protein such as a receptor protein can be bound by a specific antibody.
  • Cells expressing such a reporter protein can be captured by, for example, using antibodies bound to a solid substrate, using antibodies bound to magnetic beads, or capturing antibodies bound to the reporter protein. Many techniques for the use of antibodies as capture agents are known and can be used with the disclosed method.
  • a preferred form of cell surface protein for use as the first reporter protein is CD8 when the second reporter protein is CD4, otherwise CD4 is preferred.
  • the reporter protein can also be a protein that regulates the expression of another gene. This allows detection of expression of the reporter protein by detecting expression of the regulated gene.
  • a repressor protein can be used as the reporter protein. Inhibition of expression of the reporter protein would then result in derepression of the regulated gene.
  • This type of indirect detection allows positive detection of inhibition of the expression of the reporter protein by the functional oligonucleotide molecule.
  • One preferred form of this type of regulation is the use of an antibiotic resistance gene regulated by a repressor protein used as the reporter protein. By exposing the host cells to the antibiotic, only those cells in which expression of the reporter gene has been inhibited will grow since expression of the antibiotic resistance gene will be derepressed.
  • the second reporter protein be a protein that confers antibiotic resistance on the host cell or a cell surface protein.
  • the use of an antibiotic resistance protein is preferred in prokaryotic host cells, and the use of a cell surface protein is preferred in eukaryotic host cells.
  • the most preferred cell surface protein for use as the second reporter protein is CD4.
  • Genes encoding proteins for use as selection agents can also be inserted into the vectors. Exemplary agents are shown in Table 1. Agents can confer resistance to an antibiotic or other toxic agent to the cell, or aid in selection of cells containing functional oligonucleotide molecules.
  • the vectors can be constructed using well established recombinant DNA techniques (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, New York (1990)). It is preferred that a base vector be prepared first. Then DNA encoding an RNA molecule of interest can be inserted into this base vector to form a second base vector. A different second base vector can be constructed for each RNA molecule of interest. Finally, libraries of DNA encoding functional oligonucleotide molecules can be inserted into appropriate second base vectors. The same base vector can be easily used with any RNA molecule of interest, and the same second base vector can be used with any appropriate library of functional oligonucleotide molecules. For example, the same second base vector can be used for a library of ribozymes, a library of EGSs, and a library of antisense RNA molecules.
  • Host cells can be transformed with the disclosed vectors using any suitable means and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or detecting expression. Suitable culture conditions for host cells, such as temperature and pH, are well known. The concentration of plasmid used for cellular transfection is preferably titrated to reduce the possibility of expression in the same cell of multiple vectors encoding different functional oligonucleotide molecules.
  • Preferred prokaryotic host cells for use in the disclosed method are E. coli cells.
  • Preferred eukaryotic host cells for use in the disclosed method are monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293, Graham et al. J. Gen Virol. 36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216, [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod.
  • monkey kidney cells CVI ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci 383:44-68 (1982)); human B cells (Daudi, ATCC CCL 213); human T cells (MOLT-4, ATCC CRL 1582); and human macrophage cells (U-937, ATCC CRL 1593).
  • EGSs are short oligoribonucleotides of the general composition N Corp> 7 NACCA. If such sequences anneal to complementary sequence in a second RNA, the duplex serves as a substrate for RNase P.
  • RNase P is a ubiquitous enzyme whose normal role in cellular metabolism is 5' end maturation of transfer RNA. All characterized RNase P's are comprised of RNA and protein components. In bacteria, there is a single protein subunit and a single RNA subunit; the RNA component is catalytic. In tRNA maturation, RNase P recognizes determinants in the acceptor stem, the D loop and the non-duplexed CCA; indeed the CCA is known to form base pairs with the RNA subunit of the enzyme.
  • the EGS binds via base pairing to another RNA (the target).
  • the RNA-RNA duplex mimics the natural tRNA substrate of RNase P and recruits the enzyme, which cleaves the target RNA at the single-strand/double-strand boundary.
  • EGSs can, in theory, direct RNase P cleavage of any RNA in the cell, including mRNA.
  • the ability of RNase P to cleave RNAs other than its natural substrates serves as the underlying rationale for methodology described below.
  • the random or overlapping sequence in the EGSs for use in identifying essential or functional genes in bacteria will typically be between N ⁇ o- ⁇ 3 ; in fungi, it will typically be between N 10 - 15 , and in mammalian cells, it will typically be between N ⁇ 0 .] 8 , followed by sequence required to target the RNase P of the host cell to cleave the targeted RNA.
  • the prokaryotic RNase P this is as simple as ACCA; the structures required for the eukaryotic RNase P are more complex, and can be designed as necessary as discussed above, to create a structure similar to that of a portion of a tRNA molecule.
  • Example 1 Initial proof of principle of genome wide functional analysis using libraries of EGS sequences.
  • EGS expression must be regulatable; i.e. if EGS expression was constitutive, those cells harboring the desired sequence would die and consequently the appropriate cells would be lost upon propagation of the library.
  • regulatable promoters known in E. coli. Particularly preferred promoters initiate transcription at the site of insertion of foreign sequences (e.g. the regulatable arabinose promoter).
  • the example uses the ara C-pBAD system for conditional expression of EGS sequences. This is a tightly regulated system where transcription is repressed in the absence of arabinose and induced in the presence of low levels of arabinose.
  • Figures 2a-c shows the constructs and plasmids used in the experiments.
  • the ara c-pBAD promoter is immediately juxtaposed to an invariant A (to facilitate initiation of transcription) followed by the EGS- ACCA sequence.
  • This sequence in turn is followed by the T7e transcriptional terminator.
  • Transcripts from such constructs contain the EGS-ACCA sequence and the terminator.
  • sequence 3 ' to the EGS-ACCA does not interfere with EGS function.
  • the presence of the terminator sequence with its attendant 3' stem loop structure enhances EGS-mediated activity, presumably because the stem-loop renders the short transcripts more resistant to decay.
  • EGS sequences attached to the terminator accumulate to higher steady state levels than EGS sequence alone (i.e. those generated by cw-acting ribozymes).
  • EGS expression was assessed by RNA blot analysis of EGS sequence under a variety of conditions. As expected, no expression is observed in cells harboring the EGS plasmid if arabinose is not added to the growth media. In the presence of arabinose, EGS expression is readily detected and a high steady state level of transcript is observed following 30 minutes of induction. This level of expression is maintained for several hours.
  • a library of randomized EGSs was created by ligating the appropriate DNA insert into the pBAD plasmid to express all possible EGS sequences. The ligated plasmids were then used to transform E. coli. Sufficient independent transformants were obtained to insure that the entire library was represented (complexity of EGS sequences was approximately 4 X 10 ; independent transformants obtained was approximately 2 X 10 7 ; this number of transformants gives 95% confidence that all members of the library are represented). Following transformation, the library (each bacteria contains one plasmid, hence one EGS) was amplified via liquid growth under non-inducing conditions. Many (greater than 50) individual plasmids, were then purified and sequenced to verify that the sequence of the insert was correct and that there were no nucleotide biases in the randomized (EGS) region.
  • each active clone was then determined and compared with the published nucleotide sequence of E. coli. It was anticipated that the active EGS sequences would be complementary to transcribed regions of the genome. However, in no case was perfect complementarity (Watson-Crick) observed. These observations indicated that, among other possibilities, thirteen nucleotides of EGS sequence may contain informational content in excess of that required for biological function.
  • Active EGS sequences can be efficiently and rapidly isolated from complex libraries.
  • Example 2 Isolation and characterization of EGS sequences that when expressed cause impairment of growth or lethality.
  • Example 1 demonstrated EGS-dependent survival in the presence of certain selective agents.
  • studies were conducted to obtain EGS sequences that, when expressed, caused a loss of viability.
  • these experiments differ somewhat in design from those outlined in Example 1, i.e. there is no way to select for dead or dying cells. Accordingly, it is necessary to screen for those EGSs whose expression leads to impairment of growth.
  • the praticality of screening for lethal EGS sequences using simple replica plating depends upon the complexity of the library and the frequency of expected positives. Using the limited available data, it is possible to estimate the expected number of "positive" clones.
  • the E. coli genome has a complexity of 4.6 X 10 6 bp. If one assumes a transcription density approaching 100% and little, if any, symmetric transcription, the transcribed complexity is also 4.6 X 10 6 . Further, a reasonable estimate of essential genes is approximately 10%.
  • the effective target size for EGSs eliciting a lethal phenotype would be 4.6 X 10 bases or about 400 genes of 1 kb (not taking into consideration polarity effects and the fact that a majority of genes in E. coli are transcribed as part of operons). From the data set obtained with the second method as shown by Example 4, it is likely that only about 2% of the targetable nucleotides are "accessible", or approximately 10,000 targets distributed throughout the genome. Thus, with a truly randomized library, one would expect approximately 10,000 active EGSs.
  • the N ⁇ CC library In the case of the N ⁇ CC library only 1/16 of the possible sites would be targetable, reducing the number of active EGSs to several hundred. Given these considerations (and acknowledging the assumptions) the N ⁇ CC library, which has a complexity of 4 X 10 6 , would yield one positive clone per 10,000 clones examined.
  • Penicillin enrichment has been used for decades in microbiological research to aid in the recovery of rare mutations. The technique is based upon the fact that penicillin, or its various derivatives including ampicillin, kill only actively growing cells; cells that are not growing escape penicillin killing. The application of this enrichment strategy to isolation of lethal EGSs is fairly straightforward. Briefly, after induction of EGS expression with arabinose, those cells harboring "effective" EGSs (i.e. those targeted to essential genes) will cease to grow and thus become resistant to penicillin killing.
  • the time at which individual cells cease growing is a function of how long it takes (via turnover or dilution) for the preexisting protein of interest to decline below a viable threshold of activity. Accordingly, to enrich for lethal EGSs targeted to mRNAs encoding different essential proteins it is necessary to treat with penicillin at various times post induction (see Figure 4).
  • An enrichment experiment was performed as outlined above and, following amplification of survivors, cells recovered at various times post induction were assayed for inducer dependent lethality via replica plating. Several clones were recovered that demonstrated unambiguous growth defects only in the presence of inducer. As described above, the relevant plasmids were recovered, sequenced, and retransformed into na ⁇ ve bacteria.
  • the phenotype (slow or no growth) was shown to be due to the presence and expression of the specific EGS sequence.
  • the nucleotide sequences were compared with the E. coli genomic sequence. Again, there was not perfect (Watson-Crick) complementarity between the recovered EGSs and the published sequences.
  • N 9 ACCA, N 10 ACCA, N11AACCA and N J ACCA were prepared to explore the length requirement in vivo. Unlike the 13mer library described above, no bases in the guide sequence were held invariant. In all cases, sufficient transformants (5X complexity) were obtained to insure adequate representation.
  • N 9 and N 10 libraries To assess the activity of the N 9 and N 10 libraries, approximately 45,000 individual clones from each were assayed for their ability to form colonies in the presence or absence of EGS expression, i.e. in the presence or absence of arabinose.
  • the complexity of the N 9 library is approximately 2.6 X 10 5 ; and the complexity of the N 10 library is approximately 1 XI 0 6 . With both libraries, very few positive clones were found.
  • the extrapolated number of "active" molecules in the N 9 library was > 50 and the extrapolated number of "active" molecules in the N 10 library was approximately 100.
  • the absolute, unenriched activity of the 1 lmer library was determined by the analysis of 45,000 independent colonies. Extrapolating from the number of active (growth- inhibitory) EGS sequences in this population, the 1 lmer library contains between approximately 1 ,200 and 1 ,400 active molecules, out of a total complexity of approximately 4 x 10 6 . The increase in activity in the 1 lmer library relative to the 9 and 10m er libraries suggested that a total complementarity of 11 was the minimum to elicit significant biological activity. Furthermore, the number of "active" EGS sequences was in excess of the predicted total number of "essential” genes, but considerably below a conservative estimate of the number of total active EGSs predicted by the second method exemplified by Example 4.
  • NGR2344 Rhodothermus marinus, Rickettsia prowazekii, Ruminococcus flavefaciens, Salmonella berta, Salmonella typhimurium, Staphylococcus aureus, Streptococcus agalactiae, Streptomyces clavuligerus, Streptomyces nigrifaciens, Streptomyces phaeochromogenes, Synechocystis PCC6803, Treponema pallidum, Yersiniapestis, Zymomonas mobilis) are underway.
  • the EGSs causing cleavage of an RNA encoding an essential or functional gene is identified by comparison with the known sequence, looking for regions of complementarity by Watson-Crick base pairing.
  • a second approach relies on a direct analysis of expression pattern of RNAs in EGS -expressing cells.
  • the availability of entire genomic sequences has permitted the creation of ordered anays of the DNA sequence.
  • Such arrays can be created on traditional membranes or on miniaturized micro-chips. If total RNA from a cell population is used to probe such arrays, it is possible to obtain an expression profile of all genes simultaneously.
  • a specific mRNA will be cleaved and correspondingly its expression should be lowered in an array hybridization. This deficit in hybridization reveals the targeted sequences. As cells die or slow their growth, expression of a number of genes will change.
  • the recovered effective EGS-sequences are themselves valuable reagents for attenuating the expression of specific genes.
  • EGS sequences could be administered exogenously to bacterial populations.
  • delivery vehicles the EGSs themselves could be used as antibacterial therapeutics.
  • Vectors and a method for the identification of functional oligonucleotide molecules such as ribozymes, EGSs, and anti-sense RNA, that inhibit expression of target RNA molecules, are disclosed.
  • the method identifies functional oligonucleotide molecules by selecting for those RNA molecules that alter expression of a fusion transcript, which includes the sequence of an RNA molecule of interest, from a library of potential functional oligonucleotide molecules. Inhibition of expression of the fusion transcript prevents expression of the reporter protein. This allows inhibition of expression to be monitored by detecting expression of the reporter protein, directly or indirectly. Alternatively, expression can be increased relative to expression of the molecules in cells not including the optimal functional oligonucleotide molecule.
  • the inhibition is accomplished by interaction of a nucleic acid molecule involved in the expression of the RNA molecule of interest with a functional oligonucleotide molecule.
  • Ribozymes and EGSs result in cleavage of the fusion transcript, and antisense RNA blocks expression through hybridization to a nucleic acid molecule involved in the expression of the fusion transcript.
  • Vectors for use in functional oligonucleotide molecule assays include a first reporter gene, a second reporter gene, and a targeting gene.
  • Reporter gene 1 encodes an RNA molecule including an RNA molecule of interest and sequence encoding reporter protein A.
  • the fusion transcript includes in the 5' portion of the transcript, sequence of an RNA molecule of interest and, in the 3' region of the transcript, sequence encoding the first reporter protein.
  • the sequences are joined so that the fusion transcript encodes a fusion protein that is a fusion between the protein encoded by the sequence of the RNA molecule of interest and the reporter protein. This arrangement makes expression of the reporter protein dependent on expression of the RNA of interest.
  • Reporter gene 1 also includes expression sequences necessary for expression of the gene in appropriate host cells.
  • Reporter gene 2 encodes a different reporter protein B.
  • the vector also encodes a functional oligonucleotide molecule either specifically targeted to the RNA of interest or including a degenerate or partially
  • reporter protein A is used to assess the effect of the functional oligonucleotide molecule.
  • Expression of reporter protein B can be used both to detect transformation or transfection of the vector into cells and as a control for effects on the expression of reporter protein A that are not due to cleavage of the RNA molecule of interest.
  • the reporter genes and the targeting gene may be on separate molecules.
  • the reporter genes and the targeting gene may be on separate molecules.
  • the molecule containing the reporter genes is integrated into the host chromosome. This allows a cell strain containing appropriate reporter genes to be easily maintained and different sets of vectors encoding different libraries of functional oligonucleotide molecules to be conveniently tested against the same reporter gene.
  • reporter gene 2 is identified by detecting the presence of reporter protein B either directly or indirectly.
  • Reporter gene 2 is used to insure that the cells contain the vector and to control for any factors that could affect expression in general. Without such a control, a loss of expression of reporter gene 1 could be misinterpreted. It is not important that the level of expression of reporter gene 2 be measured. It is prefened that reporter protein B is an essential protein for the cell, such as a protein that confers antibiotic resistance or a protein that produces a required nutrient not present in culture medium, to facilitate selection.
  • Cells in which expression of the first reporter gene is altered can be identified by measuring the level of expression of reporter protein A either directly or indirectly, or by separating cells based on the expression level of reporter protein A.
  • the prefened method of detection will depend on the nature of reporter protein being used. For example, when using a reporter protein that produces a detectable signal proportionate to the level of expression, cells can be sorted or picked based on the level of signal produced. Reporter proteins such as ⁇ -galactosidase and green fluorescent protein are in this category.
  • Reporter proteins such as ⁇ -galactosidase and green fluorescent protein are in this category.
  • the cell sorting techniques described above can be used. Cells can also be sorted by FACS when using green fluorescent protein as reporter protein A since it produces a fluorescent signal.
  • the above selection process can be repeated several times, by isolating vectors from the selected cells and re-introducing them into new cells, until cells bearing a homogeneous population of plasmids can be isolated.
  • the vectors can be isolated as described below, amplified, and the sequence of the functional oligonucleotide molecule encoded in each preparation of vector can be determined.
  • Functional oligonucleotide molecules that are effective inhibitors of expression of reporter gene 1 can be identified using any suitable technique. It is prefened that the sequence of the functional oligonucleotide molecules be determined by sequencing the vectors in the selected cells. Many techniques for sequencing vector sequences from clones are known and can be used in the disclosed method. For example, Hirt supernatants of selected cells can be made and plasmids will be extracted from those cells. A prefened method for identifying the sequence of the functional oligonucleotide molecules in the isolated vectors is a single cell PCR amplification of the functional nucleotide region, followed by sequencing.
  • Another prefened method for identifying the sequence of the functional oligonucleotide molecules in the isolated vectors is to lyse the cells, extract the plasmids, amplify the plasmids in bacteria, and sequence the amplified plasmids to identify the functional oligonucleotide molecule sequence associated with the cell population.
  • Functional oligonucleotide molecules identified using the disclosed method can be used to design oligomers argeted to the same site as the functional oligonucleotide molecule. Transformation in a Single Plasmid
  • a set of the vectors encoding a first reporter gene encoding GFP as reporter protein A, a second reporter gene, and targeting gene encoding a library of EGS or ribozyme molecules as the functional oligonucleotide molecules are amplified by growing the mixed population in E. coh.
  • a fixed concentration of plasmids is complexed with an appropriate carrier (for example, lipid, calcium phosphate, DEAE dextran) and delivered to mammalian cells.
  • an appropriate carrier for example, lipid, calcium phosphate, DEAE dextran
  • the level of expression of GFP and the second reporter are measured by FACS sorting.
  • the expression of the second reporter (for example, CD4) is measured at a wavelength that does not overlap with GFP fluorescence spectrum.
  • an antibody conjugated with a fluorescent tag is used and directed against the second reporter protein to monitor the level of expression of the second reporter.
  • the antibody is incubated with the cells, excess antibody is washed off, and the fluorescence is monitored at a wavelength different from GFP.
  • the ratio of GFP expression to second reporter expression is used as a measure to determine the degree of inhibition of expression of the target sequence.
  • the cells are lysed, plasmid extracted, amplified in bacteria, and sequenced to identify the EGS or ribozyme associated with the cell population.
  • two separate plasmids are used to transform E. coli.
  • the first one encodes the fusion protein (target-GFP) and the second one encodes the second reporter and the targeting gene encoding a library of EGS or ribozymes.
  • the plasmids encoding the EGS/ribozyme library are grown in bacteria and mixed plasmids prepared as described above.
  • a fixed concentration of the mixed plasmids (each encoding a separate EGS or ribozyme) is combined with a fixed concentration of the target plasmid (encoding the target-GFP fusion protein).
  • the mixture is complexed with a commercially available preparation of lipid or calcium phosphate and transfected to cells plated in 96 wells.
  • the levels of GFP-fluorescence and the level of expression of the second reporter are measured and the ratio of GFP expression to second reporter is used to determine the efficacy of EGS or ribozyme.
  • the ratio of EGS to target can be altered to change the level of expression of the EGS or ribozyme over the target.
  • a library of DNA encoding 55 EGS sequences complementary to the CAT gene was inserted into the targeting gene of the base vector.
  • the first library Library A, encoded EGS followed by a T7 terminator.
  • the second library encoded EGS followed by a self- cleaving hammerhead ribozyme to mature 3' end of the EGS. Expression in the second library was lower, presumably due to lower stability.
  • EGSs 1 and 2 shown in Guerrier-Takeda, et al., Proc. Natl. Acad. USA 1997), previously identified functional EGS targeted to CAT RNA, show little or no activity in these assays.
  • EGS 52, EGS 36 and EGS 20 were identified as the most frequently selected EGS molecules. These same EGS molecules should be the most effective at inhibition of CAT gene expression. To test this, EGS 52, EGS 36, and EGS 20 were expressed in cells expressing a CAT gene, and the cells were challenged with chloramphenicol. The results are shown in Figures 9a and 9b . All three of the selected EGS molecules have a significant effect on chloramphenicol resistance, while cells with a control plasmid lacking any EGS exhibit chloramphenicol resistance.
  • FIG 10 further illustrates the data.
  • Each X represents an EGS complementary to a specific sequence of the mRNA transcript of the CAT gene.
  • the X's show that EGSs were recovered and that the library was represented in the experiment.
  • the same EGS was found in a number of different clones that were chosen because of their light blue or off-white color. Equally important, most of the randomly picked EGSs from the non-indicator plate were different from the active EGSs selected from the indicator plate. Most potential loci in a mRNA molecule apparently were not accessible to the action of a complementary EGS. However, specific mRNA sites were identified that are accessible to an EGS.
  • the data in Figure 10 also indicates the relative efficiency by which each EGS knocks down the expression of the targeted gene.
  • the most effective was EGS-52, which resulted in a 92% reduction of beta- galactosidase activity.
  • Three other selected EGSs (EGS-31, EGS-20, and EGS-36) reduced beta-galactosidase activity by 20% and 30%, respectively/

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Abstract

L'invention concerne deux méthodologies. Dans la première, un moyen pour identifier rapidement et efficacement des gènes fonctionnels et essentiels est prévu. Dans la deuxième, un moyen pour produire des molécules nucléiques biologiquement actives (ribozymes, séquences guides externes et antisens) qui peuvent être utilisées pour l'inactivation de gènes fonctionnels. Dans le premier procédé, une banque de séquences guides externes (EGS) à base de toutes les compositions possibles connues est préparée. Dans un mode de réalisation préféré, les EGS constituent douze ou treize mères pour cibler l'RNase bactérien pour le clivage d'un substrat. Cette banque est ajoutée à des cellules contenant les gènes à cribler, par exemple, E. coli. Les cellules dans lesquelles EGS provoque une perte de viabilité, ou d'autre phénotype, sont identifiées. La ou les EGS responsables de la perte de viabilité sont analysées, et l'information relative à la séquence résultante est utilisée pour l'identification du gène dans des séquences génomiques connues. Dans le deuxième procédé, les molécules nucléotidiques ayant une activité biologique optimale, par exemple, dirigeant le clivage d'un gène particulier par RNase P, sont rapidement identifiées au moyen d'un vecteur comprenant deux gènes reporters, le premier en phase avec le gène en question et le deuxième permettant de vérifier que le vecteur est présent dans une cellule ou facilitant la sélection de cellules contenant le vecteurs. Les cellules dans lesquelles le gène en question est clivé par la molécule oligonucléotidique fonctionnelle peuvent ensuite être identifiées en fonction du gène reporter 1. Les molécules oligonucléotidiques fonctionnelles responsables sont ensuite isolées et caractérisées. Lesdits procédés constituent des outils puissants pour l'identification de gènes essentiels dont la séquence est connue seulement comme faisant partie intégrante d'un génome à fonction inconnue, ainsi que des moyens d'identification de molécules oligonucléotidiques fonctionnelles, utiles comme réactifs diagnostiques et comme agents thérapeutiques.
PCT/US1998/024854 1997-11-21 1998-11-20 Procede d'identification et d'inhibition de molecules fonctionnelles d'acide nucleique dans des cellules WO1999027135A2 (fr)

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AU15323/99A AU732321B2 (en) 1997-11-21 1998-11-20 Method for identifying and inhibiting functional nucleic acid molecules in cells
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WO2000058457A2 (fr) * 1999-03-31 2000-10-05 Rosetta Inpharmatics, Inc. Genes de levure essentiels utilises comme cibles pour agents antifongiques, herbicides, insecticides et medicaments anti-proliferation
WO2000075370A1 (fr) * 1999-06-03 2000-12-14 Oxford Biomedica (Uk) Limited Procede de selection in vivo permettant de determiner les molecules inhibitrices d'arn
WO2001044456A2 (fr) * 1999-12-17 2001-06-21 The University Of Nottingham Modification de micro-organismes
WO2001048239A2 (fr) * 1999-12-23 2001-07-05 Xantos Biomedicine Ag Procede d'echantillonnage pour acides nucleiques
AU2008202554B2 (en) * 2001-02-23 2011-03-10 Dsm Ip Assets B.V. Novel genes encoding novel proteolytic enzymes
WO2012005982A2 (fr) * 2010-07-06 2012-01-12 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Rapporteur pour une terminaison par la arn polymérase ii

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EP1034308A1 (fr) * 1997-12-04 2000-09-13 Smithkline Beecham Corporation Procede mettant en oeuvre des sequences antisens d'expression pour produire des cellules mutantes exprimees de maniere conditionnelle
EP1034308A4 (fr) * 1997-12-04 2002-04-03 Smithkline Beecham Corp Procede mettant en oeuvre des sequences antisens d'expression pour produire des cellules mutantes exprimees de maniere conditionnelle
WO2000058457A2 (fr) * 1999-03-31 2000-10-05 Rosetta Inpharmatics, Inc. Genes de levure essentiels utilises comme cibles pour agents antifongiques, herbicides, insecticides et medicaments anti-proliferation
WO2000058457A3 (fr) * 1999-03-31 2001-01-04 Rosetta Inpharmatics Inc Genes de levure essentiels utilises comme cibles pour agents antifongiques, herbicides, insecticides et medicaments anti-proliferation
WO2000075370A1 (fr) * 1999-06-03 2000-12-14 Oxford Biomedica (Uk) Limited Procede de selection in vivo permettant de determiner les molecules inhibitrices d'arn
GB2364775A (en) * 1999-06-03 2002-02-06 Oxford Biomedica Ltd In vivo selection method for determining inhibitory RNA molecules
WO2001044456A3 (fr) * 1999-12-17 2001-12-27 Univ Nottingham Modification de micro-organismes
WO2001044456A2 (fr) * 1999-12-17 2001-06-21 The University Of Nottingham Modification de micro-organismes
WO2001048239A2 (fr) * 1999-12-23 2001-07-05 Xantos Biomedicine Ag Procede d'echantillonnage pour acides nucleiques
WO2001048239A3 (fr) * 1999-12-23 2002-08-01 Xantos Biomedicine Ag Procede d'echantillonnage pour acides nucleiques
AU2008202554B2 (en) * 2001-02-23 2011-03-10 Dsm Ip Assets B.V. Novel genes encoding novel proteolytic enzymes
AU2008202554C1 (en) * 2001-02-23 2011-07-07 Dsm Ip Assets B.V. Novel genes encoding novel proteolytic enzymes
WO2012005982A2 (fr) * 2010-07-06 2012-01-12 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Rapporteur pour une terminaison par la arn polymérase ii
WO2012005982A3 (fr) * 2010-07-06 2012-05-18 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Rapporteur pour une terminaison par la arn polymérase ii

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